Composition for use as an assay reagent

ABSTRACT

A composition for use as an assay reagent comprises a solid support comprising a member of a signal producing system and a coating of a synthetic copolymer. The synthetic copolymer comprises a first polymerized monomer comprising a pendant moiety comprising a reactive functionality or a derivative of a reactive functionality and a second polymerized monomer comprising a pendant moiety comprising at least 1 carbon atoms and at least 2 heteroatoms. In some embodiments the copolymer comprises a polyethylenic backbone comprising the pendant moiety comprising a reactive functionality or a derivative of a reactive functionality and the pendant moiety comprising at least 1 carbon atom and at least 2 heteroatoms.

BACKGROUND

The present invention relates generally to compositions useful as signalgeneration components in assays.

Dextran coating on particulate assay reagents has been employed toprovide functional groups for conjugation to moieties such as biologicalmoieties (e.g., antibodies). However, there is a significant variabilityin the chemical composition of dextran depending on the source.

There is a need for a coating having a reliable chemical composition tobe used on the surface of particles for linking various moieties to theparticles.

SUMMARY

One embodiment of the present invention is a composition that comprisesa solid support comprising a member of a signal producing system (sps)and a coating of a synthetic copolymer. The synthetic copolymercomprises a first copolymerized monomer comprising a pendant moietycomprising a reactive functionality or a derivatized reactivefunctionality and a second copolymerized monomer comprising a pendantmoiety comprising at least 1 carbon atom and at least 2 heteroatoms. Insome embodiments the pendant moiety comprising a reactive functionalityor a derivatized reactive functionality is a pendant moiety comprisingan aldehyde or an aldehyde derivative.

Another embodiment of the present invention is a method of determiningin a sample one or both of the presence and the amount of an analyte.The method comprises providing in combination in a medium the sample andthe aforementioned composition, which also comprises a member of aspecific binding pair (sbp) associated with the solid support, whereinthe member of the specific binding pair binds to the analyte or to asecond sbp member to form a complex related to the presence of theanalyte. The combination is subjected to conditions for binding of theanalyte to the composition to form a complex. The member of the signalproducing system is activated and the amount of the complex is detected.The amount of the complex is related to one or both of the presence andthe amount of analyte in the sample.

Another embodiment of the present invention is a method of determiningin a sample one or both of the presence and the amount of an analyte. Acombination is provided in a medium. The combination comprises thesample and a composition comprising a particle comprising an sps member,an sbp member that binds to the analyte or to a second sbp member toform a complex related to the presence of the analyte and a coating of acopolymer. The copolymer has the formula:

wherein: D is

(i) —COOR¹⁰ wherein R¹⁰ is H or alkyl of from 1 to 6 carbon atoms;

(ii)

wherein: w is 2-4; or

(iii) —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z

wherein:

-   -   A is O or NR¹ wherein R¹ is H or alkyl of from 1 to 6 carbon        atoms;    -   n is 1 to 10;    -   G is CHO; CH(OR⁸)₂ wherein R⁸ is alkyl of from 1 to 6 carbon        atoms; COOH or a derivative thereof; NR¹ wherein R¹ is H or        alkyl of from 1 to 6 carbon atoms and n is 1 to 10; OH; or a        member of a specific binding pair;    -   X is O or NR² wherein R² is H or alkyl of from 1 to 6 carbon        atoms;    -   Y is —(CH₂O)_(m)— wherein m is 1 to 1500; or >N^(⊕)(R³R⁴)        wherein R³ and R⁴ are independently H or alkyl of from 1 to 6        carbon atoms;    -   p is 0 to 10, being at least 1 when Y is >N^(⊕)(R³R⁴);    -   q is 0 or 1;    -   r is 0 to 10, being at least 1 when Y is >N^(⊕)(R³R⁴); and    -   Z is SO₃ ⁻; alkyl of from 1 to 6 carbon atoms;        —(CHOH)_(t)(CH₂)_(u)CH₃ wherein t is 1 to 5 and u is 0 to 10;    -   R⁵, R⁶ and R⁷ are independently H or alkyl of from 1 to 6 carbon        atoms; and    -   x and y are independently 1 to 1000.        The combination is subjected to conditions for binding of the        sbp member to the analyte or to the second sbp member to form a        complex. The sps member is activated and the amount of the        complex is detected. The amount of the complex is related to one        or both of the presence and the amount of analyte in the sample.

Another embodiment of the present invention is a copolymer of theformula:

wherein:

A is O or NR¹ wherein R¹ is H or alkyl of from 1 to 6 carbon atoms;

n is 1 to 10;

G′ is CHO; CH(OR⁸)₂ wherein R⁸ is alkyl of from 1 to 6 carbon atoms; amember of a specific binding pair;

D′ is

-   -   (i) —COOR¹⁰ wherein R¹⁰ is H or alkyl of from 1 to 6 carbon        atoms;    -   (ii)

wherein: w is 2-4; or

-   -   (iii) —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z′

wherein:

X is O or NR² wherein R² is H or alkyl of from 1 to 6 carbon atoms;

Y is —(CH₂O)_(m)— wherein m is 1 to 1500, or >N^(⊕)(R³R⁴) wherein R³ andR⁴ are independently H or alkyl of from 1 to 6 carbon atoms;

p is 0 to 10, being at least 1 when Y is >N^(⊕)(R³R⁴);

q is 0 or 1;

r is 0 to 10, being at least 1 when Y is >N^(⊕)(R³R⁴); and

Z′ is SO₃ ⁻; or alkyl of from 1 to 6 carbon atoms;

R⁵, R⁶ and R⁷ are independently H or alkyl of from 1 to 6 carbon atoms;and

x and y are independently 1 to about 1000.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of a synthesis of MAMDMA.

FIG. 2 is a tabular depiction of structures of embodiments of copolymersin accordance with the present compositions after hydrolysis of anacetal protecting group. FIG. 2 also includes the feed ratios ofhydrophilic monomers that were employed in the synthesis of thecopolymers depicted.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS Compositions

In some embodiments a copolymer of the present compositions is a randomcopolymer where one of the monomer units of the copolymer comprisesreactive functionalities for conjugation to molecules of interest suchas, for example, particles and sbp members, and the other of the monomerunits of the copolymer is functionalized to render the resultingcopolymer hydrophilic. In some embodiments, the copolymer comprises twodifferent monomer units, or three different monomer units, or fourdifferent monomer units, or five different monomer units, for example.In some embodiments, a first monomer unit comprises a reactivefunctionality or a derivative of a reactive functionality. A secondmonomer unit comprises a hydrophilic moiety. In some embodimentsadditional monomers (third, fourth and fifth monomers, for example) thatcomprise a reactive functionality or a derivative of a reactivefunctionality that is different from the reactive functionality orderivative of the reactive functionality of another of the monomers orthat comprise a hydrophilic moiety that is different from thehydrophilic moiety of another monomer may be employed.

In a random copolymer the distribution of the copolymerized monomers maybe such that at any point in the polymer chain a first copolymerizedmonomer and a second copolymerized monomer may alternate or may repeatas distinguished from block copolymers.

In some embodiments the number of each different copolymerized monomerin the copolymer is controlled during the preparation of thefunctionalized polymer by controlling the molar concentration of themonomer units that are employed in the preparation of the polymer. Thus,the number of each of the copolymerized monomers (x and y in theformulas below) is controlled in the final functionalized copolymer. Thecopolymer may be tailored, for example, to one or more of a particularsupport, to compositions comprising such supports and to the use of suchcomposition.

The term “monomer” or “monomer unit” means a molecule capable ofundergoing polymerization to form a polymer; the molecule comprises apolymerizable functionality. The number of monomer units depends on oneor more of the number of atoms in the monomer unit chain and thecomposition of the monomer unit, for example.

As mentioned above, compositions for use in preparing assay reagents orfor use as assay reagents comprise a solid support comprising an spsmember and a coating of a synthetic copolymer. The synthetic copolymercomprises at least two monomers that are copolymerized to form thepolymer. One of the copolymerized monomers comprises a polymer backboneand pendant moieties that comprise at least one reactive functionalityor at least one derivative of a reactive functionality. The other of thecopolymerized monomers comprises a hydrophilic moiety comprising atleast 1 carbon atom and at least 2 heteroatoms. In some embodiments thecopolymer comprises a polyethylenic backbone comprising the pendantmoiety comprising a reactive functionality or a derivatized reactivefunctionality and the pendant moiety comprising at least 1 carbon atomand at least 2 heteroatoms, or at least 4 carbon atoms and at least 3heteroatoms, or at least 4 carbon atoms and at least 2 heteroatoms, orat least 8 carbon atoms and at least 7 heteroatoms. The copolymerizedmonomers comprise a polymer backbone that comprises a chain of carbonatoms, from which one or more reactive functionality-containing moietiesor derivative of a reactive functionality-containing moieties arependant from the chain and one or more of the hydrophilic moieties arependant from the chain of the copolymer backbone.

In some embodiments the other of the copolymerized monomers thatcomprises a hydrophilic moiety comprises from 1 to about 15 carbon atomsand from 1 to about 10 heteroatoms. The copolymerized monomers comprisea polymer backbone that comprises a chain of carbon atoms, from whichone or more reactive functionality-containing moieties or derivative ofa reactive functionality-containing moieties are pendant from the chainand one or more of the hydrophilic moieties are pendant from the chainof the copolymer backbone.

The number of carbon atoms in the chain of the backbone of the copolymeris dependent on the number and nature of each of the copolymerizedmonomer units such as, e.g., the number of carbon atoms in thepolymerizable functionality of the monomer units, and the molecularweight of the copolymer, for example. The number of carbon atoms in thepolymerizable functionality of the monomer unit may be 2 to about 20, or2 to about 15, or 2 to about 10, or 2 to about 5, or 2 to about 4, or 2to 3, or 3 to about 20, or 3 to about 15, or 3 to about 10, or 3 toabout 5, or 3 to 4, for example. The number of carbon atoms in themonomer comprising a pendant reactive functionality-containing moiety orderivative of a reactive functionality-containing moiety (arbitrarilyreferred to herein as the first copolymerized monomer) is at least about3, or at least about 4, or at least about 5, or at least about 6, or atleast about 7, or at least about 8, or at least about 9, or at leastabout 10 and may be in the range of about 3 to about 15, or about 3 toabout 10, or about 3 to about 9, or about 3 to about 8, or about 3 toabout 7, or about 3 to about 6, or about 3 to about 5, or 3 to 4. Thenumber of the first copolymerized monomer in the copolymer is dependenton one or more of the need for binding to an sbp member and the need forbinding to the surface of a support. The number of the firstcopolymerized monomers in the copolymer is 1 to about 1,000, or 1 toabout 750, or 1 to about 500, or 1 to about 250, or 1 to about 100, or 1to about 50, or 2 to about 1,000, or 2 to about 750, or 2 to about 500,or 2 to about 250, or 2 to about 100, or 2 to about 50, or 5 to about1,000, or 5 to about 750, or 5 to about 500, or 5 to about 250, or 5 toabout 100, or 5 to about 50, or 10 to about 1,000, or to about 750, or10 to about 500, or 10 to about 250, or 10 to about 100, or 10 to about50, or 100 to about 1,000, or 100 to about 750, or 100 to about 500, or100 to about 250, for example.

The term “polymerizable functionality” refers to a portion of a monomerunit that reacts with a portion of another molecule of the monomer or aportion of a molecule of a different monomer such as, for example, amoiety that comprises one or more double or triple bonds such as, forexample, allyl groups, vinyl groups, acrylate groups, methacrylategroups, acrylamide groups and methacrylamide groups

The term “reactive functionality” is a functionality that can react witha corresponding reactive functionality on another molecule to form acovalent bond. Such reactive functionalities include, by way ofillustration and not limitation, aldehyde, carboxy, amino, imino,sulfhydryl and hydroxy, for example. In some embodiments the reactivefunctionality is an aldehyde and the first copolymerized monomercomprises an aldehyde moiety.

The term “derivative of a reactive functionality” means a moiety that isformed by the reaction of a reactive functionality with another moietythat comprises a functionality reactive with the reactive functionalitythereby forming a covalent bond linking two molecules together to formthe derivative. The derivative of a reactive functionality may comprisean acetal, a carboxy ester, an amide, an ether or a thioether, forexample. In some embodiments the derivative of a reactive functionalitymay be a reaction product of a reactive functionality with a reactivefunctionality of a member of a specific binding pair (sbp member)whereby the sbp member becomes covalently bound to the copolymer.Functionalities on the sbp member may be present naturally on the sbpmember or may be introduced synthetically into the sbp member. Suchfunctionalities include, for example, amine groups, hydroxyl groups,sulfhydryl groups and carboxyl groups. In some embodiments thederivative of a reactive functionality may be a reaction product of areactive functionality with a reactive functionality of a particlewhereby the copolymer becomes covalently bound to the particle therebyproviding a coating of the copolymer on the surface of the particle.Functionalities on the particle may be present naturally on the particleor may be introduced synthetically on the surface of the particle. Suchfunctionalities include amine groups, hydroxyl groups, azide groups andcarboxyl groups, for example. In some embodiments the derivative of areactive functionality is an aldehyde derivative.

The term “aldehyde derivative” means a moiety that is formed by thereaction of an aldehyde group with another moiety that comprises afunctionality reactive with an aldehyde group. The aldehyde derivativemay be an acetal that results from the reaction of two alcoholfunctionalities with a carbonyl oxygen of an aldehyde. The aldehydederivative may be a reaction product of an aldehyde group with a memberof a specific binding pair (sbp member) by means of reaction of thealdehyde with a functionality of the sbp member. Functionalities on thesbp member may be present naturally on the sbp member or may beintroduced synthetically into the sbp member. Such functionalitiesinclude, for example, amine groups. The reaction between an aldehydegroup and an sbp member may be by means of, for example, Schiff's baseformation between an alkyl amine or an aryl amine of the sbp member andthe aldehyde group. The reaction may be by means of reductive aminationinvolving the aldehyde group and an amine group of the sbp member. Inother embodiments, the aldehyde derivatives include, for example,acetals and bisulphite addition compounds. In some embodiments thealdehyde functionality may react with a corresponding amine group on thesurface of a particle whereby the particle becomes covalently bound tothe copolymer thereby providing a coating of the copolymer on thesurface of the particle. Functionalities on the particle may be presentnaturally on the particle or may be introduced synthetically on thesurface of the particle.

The other of the copolymerized monomers (hydrophilic monomer orotherwise referred to herein arbitrarily as the second copolymerizedmonomer) of the copolymer comprises a polymerizable functionality thatcomprises at least 1 carbon atom and at least 2 heteroatoms, or at least1 carbon atom and at least 3 heteroatoms, or at least 1 carbon atom andat least 4 carbon atoms, or at least 2 carbon atoms and at least 2heteroatoms, or at least 2 carbon atoms and at least 3 heteroatoms, orat least 2 carbon atoms and at least 4 heteroatoms, or at least 3 carbonatoms and at least 3 heteroatoms, or at least 3 carbon atoms and atleast 4 heteroatoms, or at least 4 carbon atoms and at least 3heteroatoms, or at least 4 carbon atoms and at least 4 heteroatoms, forexample. As mentioned above, the copolymerized monomers form a polymerbackbone comprising a chain of carbon atoms, from which one or morependant moieties depend from the chain. The number of carbon atoms inthe polymerizable functionality of the second monomer unit may be 2 toabout 20, or 2 to about 15, or 2 to about 10, or 2 to about 5, or 2 toabout 4, or 2 to 3, or 3 to about 20, or 3 to about 15, or 3 to about10, or 3 to about 5, or 3 to 4, for example.

In some embodiments the second copolymerized monomer of the copolymercomprises a polymerizable functionality and a pendant moiety thatcomprises from 1 to about 15 carbon atoms and from 1 to about 10heteroatoms, or from 1 to about 10 carbon atoms and from 1 to about 10heteroatoms, or from 1 to about 10 carbon atoms and from 1 to about 8heteroatoms, or from 1 to about 10 carbon atoms and from 1 to about 6heteroatoms, or from 1 to about 10 carbon atoms and from 1 to about 5heteroatoms, or from 1 to about 8 carbon atoms and from 1 to about 8heteroatoms, or from 1 to about 8 carbon atoms and from 1 to about 7heteroatoms, or from 1 to about 8 carbon atoms and from 1 to about 7heteroatoms, or from 1 to about 8 carbon atoms and from 1 to about 6heteroatoms, or from 4 to about 8 carbon atoms and from 3 to about 7heteroatoms, for example.

The number of carbon atoms of the pendant hydrophilic moiety is at least1, or at least about 2, or at least about 3, or at least about 4, or atleast about 5, or at least about 10, or at least about 20, or at leastabout 40, or at least about 60, or at least about 80, or at least about100, or at least about 150, or at least about 200, or at least about300, or at least about 400, and no more that about 2,500, or no morethan about 2,000, or no more that about 1,500, or no more that about1,100, for example. In some embodiments the number of carbon atoms ofthe pendant moiety is in the range of 1 to about 2,500, or 1 to about2,000, or 1 to about 1,500, or 1 to about 1,200, or 1 to about 1,100, or1 to about 1,000, or 1 to about 750, or 1 to about 500, or 1 to about400, or 1 to about 300, or 1 to about 200, or 1 to about 100, or 1 toabout 80, or 1 to about 60, or 1 to about 40, or 1 to about 20, or 1 toabout 10, or 2 to about 2,500, or 2 to about 2,000, or 2 to about 1,500,or 2 to about 1,200, or 2 to about 1,100, or 2 to about 1,000, or 2 toabout 750, or 2 to about 500, or 2 to about 400, or 2 to about 300, or 2to about 200, or 2 to about 100, or 2 to about 80, or 2 to about 60, or2 to about 40, or 2 to about 20, or 2 to about 10, or 3 to about 2,500,or 3 to about 2,000, or 3 to about 1,500, or 3 to about 1,200, or 3 toabout 1,100, or 3 to about 1,000, or 3 to about 750, or 3 to about 500,or 3 to about 400, or 3 to about 300, or 3 to about 200, or 3 to about100, or 3 to about 80, or 3 to about 60, or 3 to about 40, or 3 to about20, or 3 to about 10, or 4 to about 2,500, or 4 to about 2,000, or 4 toabout 1,500, or 4 to about 1,200, or 4 to about 1,100, or 4 to about1,000, or 4 to about 750, or 4 to about 500, or 4 to about 400, or 4 toabout 300, or 4 to about 200, or 4 to about 100, or 4 to about 80, or 4to about 60, or 4 to about 40, or 4 to about 20, or 4 to about 10, or 5to about 2,500, or 5 to about 2,000, or 5 to about 1,500, or 5 to about1,200, or 5 to about 1,100, or 5 to about 1,000, or 5 to about 750, or 5to about 500, or 5 to about 400, or 5 to about 300, or 5 to about 200,or 5 to about 100, or 5 to about 80, or 5 to about 60, or 5 to about 40,or 5 to about 20, or 5 to about 10, for example.

The number of molecules of the second copolymerized monomer in thecopolymer is 1 to about 1,000, or 1 to about 750, or 1 to about 500, or1 to about 250, or 1 to about 100, or 1 to about 50, or 2 to about1,000, or 2 to about 750, or 2 to about 500, or 2 to about 250, or 2 toabout 100, or 2 to about 50, or 5 to about 1,000, or 5 to about 750, or5 to about 500, or 5 to about 250, or 5 to about 100, or 5 to about 50,or 10 to about 1,000, or to about 750, or 10 to about 500, or 10 toabout 250, or 10 to about 100, or 10 to about 50, or 100 to about 1,000,or 100 to about 750, or 100 to about 500, or 100 to about 250, forexample.

The number of heteroatoms of the pendant hydrophilic moiety of thesecond copolymerized monomer comprising at least 1 carbon atom and atleast 2 heteroatoms is that which is sufficient to render the copolymerhydrophilic. The term “hydrophilic” refers to a property of a moleculethat is polar and thus prefers neutral molecules or polar molecules andprefers polar solvents. Hydrophilic molecules have an affinity for otherhydrophilic moieties compared to hydrophobic moieties. The number ofheteroatoms is dependent on such factors as the number of carbon atomsin the pendant moiety, the nature of the support and the nature of analdehyde derivative, if present, for example.

The number of heteroatoms of the pendant hydrophilic moiety is at leastabout 2, or at least about 3, or at least about 4, or at least about 5,or at least about 6, or at least about 7, or at least about, or at leastabout 8, or at least about 9, or at least about 10, or at least about20, or at least about 30, or at least about 40, or at least about 50, orat least about 60, or at least about 70, or at least about 80, or atleast about 90, or at least about 100, or at least about 150, and nomore that about 1,500, or no more that about 1,200, or no more thanabout 1,100, or no more that about 1,000, for example. In someembodiments the number of heteroatoms is in the range of 2 to about1,500, or 2 to about 1,250, or 2 to about 1,000, or 2 to about 750, or 2to about 500, or about 2 to about 250, or 2 to about 100, or 2 to about80, or 2 to about 60, or 2 to about 40, or 2 to about 20, or 2 to about10, or 3 to about 1,500, or 3 to about 1,250, or 3 to about 1,000, or 3to about 750, or 3 to about 500, or about 3 to about 250, or 3 to about100, or 3 to about 80, or 3 to about 60, or 3 to about 40, or 3 to about20, or 3 to about 10, or 5 to about 1,500, or 5 to about 1,250, or 5 toabout 1,000, or 5 to about 750, or 5 to about 500, or about 5 to about250, or 5 to about 100, or 5 to about 80, or 5 to about 60, or 5 toabout 40, or 5 to about 20, or 5 to about 10, or 7 to about 1,500, or 7to about 1,250, or 7 to about 1,000, or 7 to about 750, or 7 to about500, or about 7 to about 250, or 7 to about 100, or 7 to about 80, or 7to about 60, or 7 to about 40, or 7 to about 20, or 7 to about 10, forexample.

The hydrophilicity of the copolymer may be controlled by the number ofheteroatoms (and functional groups comprising such heteroatoms) in thecopolymer. The number of heteroatoms in the copolymer depends on thenumber of heteroatoms in each of the second copolymerized monomer andalso on the number of copolymerized monomers comprising the heteroatomscompared with the number of copolymerized monomers that comprise apendent moiety comprising a reactive functionality or a derivative of areactive functionality. In some embodiments there is 1 heteroatom perabout 3 carbon atoms, or 1 heteroatom per about 6 carbon atoms, or 1heteroatom per about 10 carbon atoms, or 1 heteroatom per about 12carbon atoms, or 1 heteroatom per about 15 carbon atoms, or 2heteroatoms per 1 carbon atom, or 2 heteroatoms per about 2 carbonatoms, or 2 heteroatoms per about 3 carbon atoms, or 2 heteroatoms perabout 4 carbon atoms, or 2 heteroatoms per about 6 carbon atoms, or 2heteroatoms per about 10 carbon atoms, or 2 heteroatoms per about 12carbon atoms, or 2 heteroatoms per about 15 carbon atoms, or 3heteroatoms per about 4 carbon atoms, or 3 heteroatoms per about 6carbon atoms, or 3 heteroatoms per about 10 carbon atoms, or 3heteroatoms per about 12 carbon atoms, or 3 heteroatoms per about 15carbon atoms, or 4 heteroatoms per about 4 carbon atoms, or 4heteroatoms per about 6 carbon atoms, or 4 heteroatoms per about 10carbon atoms, or 4 heteroatoms per about 12 carbon atoms, or 4heteroatoms per about 15 carbon atoms, or 5 heteroatoms per about 4carbon atoms, or 5 heteroatoms per about 6 carbon atoms, or 5heteroatoms per about 10 carbon atoms, or 5 heteroatoms per about 12carbon atoms, or 5 heteroatoms per about 15 carbon atoms, or 6heteroatoms per about 5 carbon atoms, or 6 heteroatoms per about 6carbon atoms, or 6 heteroatoms per about 10 carbon atoms, or 6heteroatoms per about 12 carbon atoms, or 6 heteroatoms per about 15carbon atoms, or 7 heteroatoms per about 7 carbon atoms, or 7heteroatoms per about 8 carbon atoms, or 7 heteroatoms per about 10carbon atoms, or 7 heteroatoms per about 12 carbon atoms, or 7heteroatoms per about 15 carbon atoms, for example.

In some embodiments the pendant moiety of the second polymerized monomer(hydrophilic monomer) may comprise at least 1 carbon atom and at least 2heteroatoms, or at least 1 carbon atom and at least 3 heteroatoms, or atleast 1 carbon atom and at least 4 carbon atoms, or at least 2 carbonatoms and at least 2 heteroatoms, or at least 2 carbon atoms and atleast 3 heteroatoms, or at least 2 carbon atoms and at least 4heteroatoms, or at least 3 carbon atoms and at least 3 heteroatoms, orat least 3 carbon atoms and at least 4 heteroatoms, or at least 4 carbonatoms and at least 2 heteroatoms, or at least 4 carbon atoms and atleast 3 heteroatoms, or at least 4 carbon atoms and at least 4heteroatoms, or at least 4 carbon atoms and at least 5 heteroatoms, orat least 5 carbon atoms and at least 2 heteroatoms, or at least 5 carbonatoms and at least 3 heteroatoms, or at least 5 carbon atoms and atleast 4 heteroatoms, or at least 5 carbon atoms and at least 5heteroatoms, or at least 5 carbon atoms and at least 6 heteroatoms, orat least 6 carbon atoms and at least 2 heteroatoms, or at least 6 carbonatoms and at least 3 heteroatoms, or at least 6 carbon atoms and atleast 4 heteroatoms, or at least 6 carbon atoms and at least 5heteroatoms, or at least 6 carbon atoms and at least 6 heteroatoms, orat least 6 carbon atoms and at least 7 heteroatoms, or at least 7 carbonatoms and at least 2 heteroatoms, or at least 7 carbon atoms and atleast 3 heteroatoms, or at least 7 carbon atoms and at least 4heteroatoms, or at least 7 carbon atoms and at least 5 heteroatoms, orat least 7 carbon atoms and at least 6 heteroatoms, or at least 7 carbonatoms and at least 7 heteroatoms, or at least 7 carbon atoms and atleast 8 heteroatoms, or at least 8 carbon atoms and at least 2heteroatoms, or at least 8 carbon atoms and at least 3 heteroatoms, orat least 8 carbon atoms and at least 4 heteroatoms, or at least 8 carbonatoms and at least 5 heteroatoms, or at least 8 carbon atoms and atleast 6 heteroatoms, or at least 8 carbon atoms and at least 7heteroatoms, or at least 8 carbon atoms and at least 8 heteroatoms, orat least 8 carbon atoms and at least 9 heteroatoms, for example.

The number of copolymerized monomers comprising the heteroatoms comparedwith the number of polymerized monomers that comprise a pendent moietycomprising a reactive functionality or a derivative of a reactivefunctionality may be controlled by the feed ratio of the monomers duringpolymerization. Accordingly, during a polymerization the feed ratio ofthe monomer comprising the pendant moiety comprising a reactivefunctionality or a derivative of a reactive functionality (firstcopolymerizable monomer) may be increased over that of the hydrophilicmonomer (second copolymerizable monomer). The ratio of the secondcopolymerizable monomer to the first copolymerizable monomer during apolymerization may be 1:1, or 1:1.5, or 1:2, or 1:2.5, or 1:3, or 1:3.5,or 1:4, or 1:4.5, or 1:5, or 1:5.5, or 1:6, for example. This feed ratiocontrols the value of x and y in the formulas below. In addition, theratio of the second copolymerizable monomer to the first copolymerizablemonomer can be adjusted to control the amount of signal obtained from asolid support comprising a member of a signal producing system (sps) anda coating of the synthetic copolymer. By having more of the firstcopolymerizable monomer comprising a pendant moiety comprising areactive functionality, one can obtain a greater amount of derivatizedreactive functionality on the solid support. In this manner, one canbalance the hydrophilic properties of the resulting solid support withcoated synthetic copolymer and the number of derivatized reactivefunctionalities to accommodate a wide variety of assays and signalproducing systems.

The heteroatoms of the pendant hydrophilic moiety include, for example,oxygen, sulfur, nitrogen and phosphorus, and combinations of oxygen,sulfur, nitrogen and phosphorus. The heteroatoms may be present incombination with other one another or with other atoms such as, forexample, one or more of hydrogen and carbon, in the form of one or morehydrophilic groups. In some embodiments, oxygen may be present as oxo oroxy bonded to one or more of hydrogen, carbon, sulfur, nitrogen andphosphorous; nitrogen may be bonded to one or more of hydrogen, carbon,oxygen, sulfur and phosphorus such as, for example, an azo, cyano,isocyano, nitro, nitroso, amido or amino group; sulfur is analogous tooxygen as discussed above; phosphorous may be bonded to one or more ofhydrogen, carbon, sulfur, oxygen and nitrogen, such as, for example, aphosphonate or phosphate mono- or diester group.

The number of hydrophilic groups in the pendant hydrophilic moiety maybe 2 to about 1,500, or 2 to about 1,250, or 2 to about 1,000, or 2 toabout 750, or 2 to about 500, or 2 to about 250, or 2 to about 125, or 2to about 100, or 2 to about 80, or 2 to about 60, or 2 to about 40, or 2to about 20, or 2 to about 10, or 2 to about 5, or 3 to about 1,500, or3 to about 1,250, or 3 to about 1,000, or 3 to about 750, or 3 to about500, or 3 to about 250, or 3 to about 125, or 3 to about 100, or 3 toabout 80, or 3 to about 60, or 3 to about 40, or 3 to about 20, or 3 toabout 10, or 3 to about 5, or 4 to about 1,500, or 4 to about 1,250, or4 to about 1,000, or 4 to about 750, or 4 to about 500, or 4 to about250, or 4 to about 125, or 4 to about 100, or 4 to about 80, or 4 toabout 60, or 4 to about 40, or 4 to about 20, or 4 to about 10, or 4 to5; the hydrophilic groups may be independently one or more of the groupsmentioned below.

In some embodiments, the hydrophilic group or groups of the pendanthydrophilic moiety, by way of illustration and not limitation, may beselected from an amine group (a primary, secondary, tertiary orquaternary amine), an amide group, a hydroxyl group, an ester group, anether group, a polyether group (e.g., a polyoxyethylene group and apolyoxypropylene group), an epoxide group, a thioether group, apolythioether group, a sulfate group, a sulfite group, a phosphategroup, a phosphite group, a phosphatidylcholine group, a betaine group,a sulfobetaine group, a nitrile group, an isonitrile group, a cyanategroup, an isocyanate group, a thiocyanate group, an isothiocyanategroup, an azide group, a thiol group, a thiolate group, a sulfide group,a sulfinate group, a sulfonate group, a phenolate group, a carbonylgroup, a carboxylate group, a phosphine group, a phosphine oxide group,a phosphonic acid group and a phosphoramide group, as well ascombinations and mixtures of such groups. In some embodiments, thehydrophilic group or groups, by way of illustration and not limitation,may be selected from the groups consisting of amine groups (primary,secondary, tertiary or quaternary amines), amide groups, hydroxylgroups, ester groups, ether groups, polyether groups, thioether groups,sulfate groups, sulfite groups, phosphate groups, phosphite groups,phosphatidylcholine groups, betaine groups and sulfobetaine groups.

In some embodiments the molecular weight (Daltons) (Da) of the copolymeris about 300 to about 10,000,000 or more, or about 500 to about10,000,000, or about 1,000 to about 10,000,000, or about 10,000 to about10,000,000, or about 100,000 to about 10,000,000, or 300 to about5,000,000 or more, or about 500 to about 5,000,000, or about 1,000 toabout 5,000,000, or about 10,000 to about 5,000,000, or about 100,000 toabout 5,000,000, or 300 to about 1,000,000 or more, or about 500 toabout 1,000,000, or about 1,000 to about 1,000,000, or about 10,000 toabout 1,000,000, or about 100,000 to about 1,000,000, or about 100 toabout 750,000, or about 500 to about 750,000, or about 1,000 to about750,000, or about 10,000 to about 750,000, or about 100,000 to about750,000, or about 100 to about 500,000, or about 200 to about 500,000,or about 1,000 to about 500,000, or about 10,000 to about 500,000, orabout 100,000 to about 500,000, for example.

In some embodiments the copolymer comprises a polyethylenic backbonefrom which depend one or more reactive functionality-containing moietiesor derivative of reactive functionality-containing such as, for example,one or more aldehyde-containing moieties or aldehydederivative-containing moieties and one or more moieties comprising atleast 1 carbon atom and at least 2 heteroatoms. The polyethylenicbackbone comprises a linear chain of ethylenic groups, i.e., —(CHR—CHR)—groups (where R is alkyl or hydrogen) formed from monomers comprisingdouble bonds. Other types of polymer backbones are also included anddepend on the nature of the monomers. The monomers from which thecopolymer is formed include, by way of example and not limitation, vinylmonomers, allylic monomers, olefins, and any small molecules containingat least one degree of unsaturation, and mixtures or two or more of theabove monomers wherein the polymerizable functionality is acarbon-carbon double bond or a carbon-carbon triple bond. Besides thepolymerizable functionality the monomer also comprises an appropriatesubstitution of either a reactive functionality or a derivative of areactive functionality or at least 1 carbon atom and at least 2heteroatoms. Classes of vinyl monomers include, but are not limited to,methacrylic acid, methacrylates, methacrylamide, N- andN,N-disubstituted methacrylamides, vinyl aromatic monomers, vinylhalides, vinyl esters of carboxylic acids (e.g., vinyl acetate),ethylene oxide acrylates, diacrylates, and dimethacrylates.

Examples of methacrylates include methacrylates appropriatelysubstituted with a pendant moiety in accordance with present embodimentswherein the methacrylates include, by way of illustration and notlimitation, methyl methacrylate, ethyl methacrylate, propylmethacrylate, isopropyl methacrylate, n-butyl methacrylate, iso-butylmethacrylate, and tert-butyl methacrylate, for example. Examples ofvinyl aromatic monomers that may be used include, but are not limitedto, appropriately substituted styrene, styrene-butadiene,p-chloromethylstyrene and divinyl benzene, for example. Vinyl halidesthat may be used include, but are not limited to, appropriatelysubstituted vinyl chloride and vinylidene fluoride. Vinyl esters ofcarboxylic acids that may be used include, but are not limited to,appropriately substituted vinyl acetate, vinyl butyrate, vinyl3,4-dimethoxybenzoate, vinyl malate and vinyl benzoate.

The copolymer is synthesized according to standard polymer chemistry forthe synthesis of random copolymers using the appropriate monomeric unitsas identified above. In some embodiments, monomer units comprising oneor more polymerizable functionalities may be combined in a singlepolymerization step. As mentioned above, in this latter polymerizationapproach, the number of each of the copolymerized monomers of thecopolymer may be controlled by controlling the molar concentration ofthe monomer units.

The random copolymers may be prepared by any polymerization techniquefor the preparation of random copolymers. Polymerization techniquesinclude, for example, radical polymerization, atom transfer radicalpolymerization, reversible addition fragmentation and chain transferpolymerization, nitroxide mediated polymerization, and so forth. Theconditions for the polymerization such as, for example, temperature,reaction medium, pH, duration, and the order of addition of the reagentsare dependent on one or more of the type of polymerization employed, thenature of the monomer reagents including any polymerizable functionalityemployed and the nature of any catalyst employed, for example. Suchconditions are generally known since the types of polymerizationtechniques that can be used are well-known in the art.

As mentioned above, a composition in accordance with some of the presentembodiments comprises a solid support comprising an sps member and acoating of the synthetic copolymer. The solid support may be comprisedof an organic or inorganic, water insoluble material, which may betransparent or partially transparent. The solid support has a surfacethat is hydrophobic and can have any of a number of shapes such as, forexample, particulate, including beads and particles, film, membrane,tube, well, strip, rod, and planar surfaces such as, e.g., plate.Depending on the type of assay, the solid support may or may not besuspendable in the medium in which it is employed. Examples of asuspendable solid support include polymeric materials such as latexparticles and magnetic particles. Other solid support compositionsinclude polymers, such as poly (vinyl chloride), polyacrylamide,polyacrylate, polyethylene, polypropylene, poly-(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, andpoly(vinyl butyrate), for example; either used by themselves or inconjunction with other materials.

In some embodiments the solid support is a particle. The particlesgenerally have an average diameter of about 0.02 to about 100 microns,or about 0.05 to about 100 microns, or about 0.1 to about 100 microns,or about 0.5 to about 100 microns, or about 0.02 to about 50 microns, orabout 0.05 to about 50 microns, or about 0.1 to about 50 microns, orabout 0.5 to about 50 microns, or about 0.02 to about 20 microns, orabout 0.05 to about 20 microns, or about 0.1 to about 20 microns, orabout 0.5 to about 20 microns, for example. In some embodiments, theparticles have an average diameter from about 0.05 microns to about 20microns or from about 0.3 microns to about 10 microns, or about 0.3microns to about 5 microns, for example. In some embodiments, theparticles are latex particles or chrome particles.

A latex particle is a particulate water suspendable, water insolublepolymeric material. In some embodiments the latex is a substitutedpolyethylene such as polystyrene-butadiene, polyacrylamide polystyrene,polystyrene with amino groups, poly-acrylic acid, polymethacrylic acid,acrylonitrile-butadiene, styrene copolymers, polyvinyl acetate-acrylate,polyvinyl pyridine, vinyl-chloride acrylate copolymers, and the like.

Polymeric particles can be formed from addition or condensationpolymers. The particles will be readily dispersible in an aqueous mediumand can be functionalizable so as to permit conjugation to one or moreof a copolymer as described herein, a member of a signal producingsystem (sps), and a member of a specific binding pair (sbp), forexample. The particles can also be derived from naturally occurringmaterials, naturally occurring materials that are syntheticallymodified, and synthetic materials. In some embodiments the particleshave, either naturally occurring or synthetically introduced, a reactivefunctionality such as, for example, amine groups, which are reactivewith a corresponding reactive functionality of the copolymer such as,for example, aldehyde groups.

As mentioned above, the copolymer is a coating on the support. Coatingthe support with the copolymer may be accomplished in a number of ways.The copolymer may be attached to the surface of the support covalently.In some embodiments, covalent attachment may be accomplished by reactionof some of the reactive functionalities such as, for example, aldehydegroups, of the copolymer with a functionality on the surface of thesupport in a manner similar to that discussed above with regard to thealdehyde derivative. As mentioned above, in some embodiments, dependingon the nature of the support, suitable functionalities may be alreadypresent on the surface of the support or they may be syntheticallyintroduced on the surface. The remaining reactive functionalities suchas, for example, aldehyde groups, are available for reaction with asuitably functionalized sbp member, for example.

In some embodiments, the amount of the copolymer coated on the supportis dependent on one or more of the nature of the support, the nature ofthe copolymer, the nature of the sps member, whether attachment of thecopolymer to the support is by virtue of the aldehyde bearing site, andwhether an sbp member is attached to the aldehyde bearing site, forexample. In some embodiments the amount (percent by weight) of copolymercoated on the support is about 0.1 to about 10%, or about 0.1 to about9%, or about 0.1 to about 8%, or about 0.1 to about 7%, or about 0.1 toabout 6%, or about 0.1 to about 5%, or about 0.1 to about 4%, or about0.1 to about 3%, or about 0.1 to about 2%, or about 0.1 to about 1%, orabout 0.1 to about 0.5%, or about 1 to about 10%, or about 1 to about9%, or about 1 to about 8%, or about 1 to about 7%, or about 1 to about6%, or about 1 to about 5%, or about 1 to about 4%, or about 1 to about3%, or about 1 to about 2%, or about 0.05 to about 0.5%, or about 0.06to about 0.5%, or about 0.07 to about 0.5%, or about 0.08 to about 0.5%,or about 0.09 to about 0.5%, or about 0.1 to about 0.5%, or about 0.05to about 0.4%, or about 0.06 to about 0.4%, or about 0.07 to about 0.4%,or about 0.08 to about 0.4%, or about 0.09 to about 0.4%, or about 0.1to about 0.4%, or about 0.05 to about 0.3%, or about 0.06 to about 0.3%,or about 0.07 to about 0.3%, or about 0.08 to about 0.3%, or about 0.09to about 0.3%, or about 0.1 to about 0.3%, or about 0.05 to about 0.2%,or about 0.06 to about 0.2%, or about 0.07 to about 0.2%, or about 0.08to about 0.2%, or about 0.09 to about 0.2%, or about 0.1 to about 0.2%,for example.

The selection of a copolymer coating for a particle comprising a memberof a signal producing system depends on one or more of a number offactors such as, for example, the type of assay, the nature of themember of the signal producing system, the expected concentration rangeof the analyte, the physical characteristics and origin of the antibody,the variation in effective antibody coating density, the pH of the finalreaction mixture, and the ionic strength of the final reaction mixture.Depending on such factors, one copolymer coating may be preferred overanother copolymer coating in any particular application. For example,the effective antibody coating density may be optimal for a signalproducing system when a particular antibody is conjugated to aparticular copolymer coating. The amount of signal may be controlled,for example, by adjusting the feed ratios of the first and secondpolymerizable monomers during copolymerization.

As mentioned above, the composition also comprises an sps member. Thenature of the sps member depends on the type of assay in whichembodiments of the present compositions may be employed. Such assaysinclude, for example, immunoprecipitin and agglutination methods andcorresponding light scattering techniques such as, e.g., nephelometryand turbidimetry, for the detection of antibody complexes; and labeledassays (e.g., labeled immunoassays) such as, for example, inducedluminescence (luminescent oxygen channeling) assays, fluorescent oxygenchanneling assays, enzyme-labeled assays, fluorescence polarizationassays, radio-labeled assays and inhibition assays.

The sps member may be a label, which is part of a signal producingsystem. The nature of the label is dependent on the particular assayformat as discussed above. A signal producing system may include one ormore components, at least one component being a detectable label, whichgenerates a detectable signal that relates to one or both of the amountof bound and unbound label, i.e. the amount of label bound or not boundto analyte being detected or to an agent that reflects the amount of theanalyte to be detected. The label is any molecule that produces or canbe induced to produce a signal, and may be, for example, a fluorescer,radiolabel, enzyme, chemiluminescer or photosensitizer. Thus, the signalis detected and/or measured by detecting enzyme activity, luminescence,light absorbance or radioactivity, for example, as the case may be.

Suitable labels include, by way of illustration and not limitation,enzymes such as alkaline phosphatase, glucose-6-phosphate dehydrogenase(“G6PDH”) and horseradish peroxidase; ribozyme; a substrate for areplicase such as QB replicase; promoters; dyes; fluorescers, such asfluorescein, isothiocyanate, rhodamine compounds, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine;complexes such as those prepared from CdSe and ZnS present insemiconductor nanocrystals known as Quantum dots; chemiluminescers suchas isoluminol; sensitizers including photosensitizers; coenzymes; enzymesubstrates; radiolabels such as ¹²⁵I, ¹³¹I, ¹⁴C, ³H, ⁵⁷Co and ⁷⁵Se;particles such as latex particles, carbon particles, metal particlesincluding magnetic particles, e.g., chromium dioxide (CrO₂) particles,and the like; metal sols; crystallite; liposomes; nucleotides and cells,for example, which may be further labeled with a dye, catalyst or otherdetectable group.

The label can directly produce a signal and, therefore, additionalcomponents are not required to produce a signal. Numerous organicmolecules, for example fluorescers, are able to absorb ultraviolet andvisible light, where the light absorption transfers energy to thesemolecules and elevates them to an excited energy state. This absorbedenergy is then dissipated by emission of light at a second wavelength.Other labels that directly produce a signal include radioactive isotopesand dyes.

Alternately, the label may need other components to produce a signal,and the signal producing system would then include all the componentsrequired to produce a measurable signal. Such other components mayinclude, for example, substrates, coenzymes, enhancers, additionalenzymes, substances that react with enzymic products, catalysts,activators, cofactors, inhibitors, scavengers, metal ions, and aspecific binding substance required for binding of signal generatingsubstances.

As mentioned above, in the present compositions the sps member isassociated with the support. The manner of association of the sps memberwith the solid support depends on one or more of the nature of thesupport, the nature of the sps member, the surface area and porosity ofthe support and the nature of any solvent employed, for example. Theassociation may be by adsorption of the sps member by the support,covalent bonding of the sps member to the support, dissolution ordispersion of the sps member in the solid support, non-covalent bondingof the sps member to the support by means of binding pair members (e.g.,avidin-biotin and digoxin-antibody for digoxin), for example. In thismanner the sps member is “associated with” the solid support.

As used herein, the phrase “associated with” includes covalent bindingof one moiety to another moiety either by a direct bond or through aspacer group, non-covalent binding of one moiety to another moietyeither directly or by means of specific binding pair members bound tothe moieties, incorporation of one moiety into another moiety such as bydissolving one moiety in another moiety or by synthesis, and coating onemoiety on another moiety, for example.

Association of an sps member such as, for example, a sensitizer or achemiluminescent compound, with latex particles may involveincorporation during formation of the particles by polymerization, orincorporation into preformed particles, e.g., by non-covalentdissolution into the particles, for example. In some approaches asolution of the sps member is employed. Solvents that may be utilizedinclude, for example, alcohols, including, e.g., ethanol, ethoxyethanol,methoxyethanol, ethylene glycol and benzyl alcohol; amides such as,e.g., dimethyl formamide, formamide, acetamide and tetramethyl urea;sulfoxides such as, e.g., dimethyl sulfoxide and sulfolane; and etherssuch as, e.g., carbitol, ethyl carbitol and dimethoxy ethane; and water;and mixtures of two or more of the above. The use of solvents havinghigh boiling points in which the particles are insoluble permits the useof elevated temperatures to facilitate dissolution of the compounds intothe particles and are particularly suitable. The solvents may be usedsingly or in combination. A solvent should be selected that does notinterfere with the signal producing ability of the sps members becauseof their intrinsic properties or ability to be removed from theparticles. In some embodiments aromatic solvents may be employed suchas, for example, dibutylphthalate, benzonitrile, naphthonitrile,dioctylterephthalate, dichlorobenzene, diphenylether anddimethoxybenzene.

Generally, the temperature employed during the procedure is chosen tomaximize the amount of signal from the sps member particles with theproviso that the particles should not melt or become aggregated at theselected temperature. In some embodiments, elevated temperatures areemployed. The temperatures for the procedure may range from about 20° C.to about 200° C., or from about 50° C. to about 170° C. It has beenobserved that some compounds that are nearly insoluble at roomtemperature are soluble in, for example, low molecular weight alcohols,such as ethanol and ethylene glycol, for example, at elevatedtemperatures. Carboxylated modified latex particles have been shown totolerate low molecular weight alcohols at such temperatures.

In some embodiments of the present compositions, the sps member isselected from the group consisting of sensitizers, includingphotosensitizers, and chemiluminescent compounds as discussed more fullyhereinbelow.

A chemiluminescent compound (chemiluminescer) is a compound that ischemically activatable and, as a result of such activation, emits lightat a certain wavelength. Examples of chemiluminescers, by way ofillustration and not limitation, include olefins capable of reactingwith singlet oxygen or a peroxide to form hydroperoxides or dioxetanes,which can decompose to ketones or carboxylic acid derivatives; stabledioxetanes which can decompose by the action of light; acetylenes whichcan react with singlet oxygen to form diketones; hydrazones orhydrazides that can form azo compounds or azo carbonyls such as luminol;and aromatic compounds that can form endoperoxides, for example. As aconsequence of the activation reaction, the chemiluminescers directly orindirectly cause the emission of light.

A sensitizer is a molecule, usually a compound, for generation of areactive intermediate such as, for example, singlet oxygen, foractivation of a chemiluminescent compound. In some embodiments of thepresent compositions, the sensitizer is a photosensitizer. Othersensitizers that can be chemi-activated (by, e.g., enzymes and metalsalts) include, by way of example and not limitation, other substancesand compositions that can produce singlet oxygen with or, lesspreferably, without activation by an external light source. For example,certain compounds have been shown to catalyze the conversion of hydrogenperoxide to singlet oxygen and water. Also included within the scope ofphotosensitizers are compounds that are not true sensitizers but whichon excitation by heat, light, ionizing radiation, or chemical activationwill release a molecule of singlet oxygen. The best known members ofthis class of compounds include the endoperoxides such as1,4-biscarboxyethyl-1,4-naphthalene endoperoxide,9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption of light bythese compounds releases singlet oxygen.

A photosensitizer is a sensitizer for activation of a photoactivecompound, for example, by generation of singlet oxygen by excitationwith light. The photosensitizers are photoactivatable and include, e.g.,dyes and aromatic compounds, and are usually compounds comprised ofcovalently bonded atoms, usually with multiple conjugated double ortriple bonds. The compounds should absorb light in the wavelength rangeof 200 to 1,100 nm, or 300 to 1,000 nm, or 450 to 950 nm, with anextinction coefficient at its absorbance maximum greater than 500 M⁻¹cm⁻¹, or greater than 5,000 M⁻¹ cm⁻¹, or greater than 50,000 M⁻¹ cm⁻¹,at the excitation wavelength. Photosensitizers should be relativelyphotostable and, preferably, not react efficiently with singlet oxygen.Examples of photosensitizers, by way of illustration and not limitation,include acetone, benzophenone, 9-thioxanthone, eosin,9,10-dibromoanthracene, methylene blue, metallo-porphyrins, such ashematoporphyrin, phthalocyanines, chlorophylls, rose bengal, andbuckminsterfullerene, for example, and derivatives of these compounds.

Examples of chemiluminescent compounds and photosensitizers that may beutilized in embodiments of the present compositions are set forth inU.S. Pat. No. 5,340,716 (Ullman, et al.), the relevant portions of whichdisclosure are incorporated herein by reference.

In some embodiments the pendant moiety of the polymerized monomer (firstpolymerized monomer) that comprises an aldehyde or an aldehydederivative is

—C(O)-A-(CH₂)_(n)-G

wherein:

A is O or NR¹ wherein R¹ is H or alkyl of from 1 to 6, or 1 to 5, or 1to 4, or 1 to 3, or 1 to 2, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3,or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 6, or 4 to 5, or 5 to 6 carbonatoms, for example;

n is 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, 1to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7,or 2 to 6, or 2 to 5, 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, forexample; and

G is CHO; CH(OR⁸)₂ wherein R⁸ is alkyl of from 1 to 6, or 1 to 5, or 1to 4, or 1 to 3, or 1 to 2, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3,or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 6, or 4 to 5, or 5 to 6 carbonatoms, for example; COOH or a derivative there of such as, e.g., anester or an amide; NR¹ wherein R¹ is H or alkyl of from 1 to 6 carbonatoms; a member of a specific binding pair.

In some embodiments the pendant moiety of the polymerized monomer thatcomprises at least 1 carbon atom and at least 2 heteroatoms has theformula:

(i) —COOR¹⁰ wherein R¹⁰ is H or alkyl of from 1 to 6, or 1 to 5, or 1 to4, or 1 to 3, or 1 to 2, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or3 to 6, or 3 to 5, or 3 to 4, or 4 to 6, or 4 to 5, or 5 to 6 carbonatoms, for example;

(ii)

wherein: w is 2-4, or 2-3, or 3-4, or 2, or 3, or 4; or

(iii) —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z

wherein:

X is O or NR² wherein R² is H or alkyl of from 1 to 6, or 1 to 5, or 1to 4, or 1 to 3, or 1 to 2, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3,or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 6, or 4 to 5, or 5 to 6 carbonatoms, for example;

Y is —(CH₂O)_(m)— wherein m is 1 to 100, or 2 to 100, or 3 to 100, or 4to 100, or 1 to 90, or 1 to 80, or 1 to 70, or 1 to 60, or 1 to 50, 1 to40, or 1 to 30, or 1 to 20, or 1 to 10, or 1 to 5, or 5 to 100, or 5 to90, or 5 to 80, or 5 to 70, or 5 to 60, or 5 to 50, 5 to 40, or 5 to 30,or 5 to 20, or 5 to 10, or 10 to 100, or 10 to 90, or 10 to 80, or 10 to70, or 10 to 60, or 10 to 50, or 10 to 40, or 10 to 30, or 10 to 20, forexample; or >N^(⊕)(R³R⁴) wherein R³ and R⁴ are independently H or alkylof from 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 6,or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 6, or 3 to 5, or 3 to 4, or 4to 6, or 4 to 5, or 5 to 6 carbon atoms, for example;

p is 0 to 10, or 0 to 9, or 0 to 8, or 0 to 7, or 0 to 6, or 0 to 5, or0 to 4, or 0 to 3, or 0 to 2, or 0 to 1, or 1 to 10, or 1 to 9, or 1 to8, or 1 to 7, or 1 to 6, or 1 to 5, 1 to 4, or 1 to 3, or 1 to 2, or 2to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, 2 to 4, or2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8,or 8 to 10, or 8 to 9, or 9 to 10, for example, being at least 1 when Yis >N^(⊕)(R³R⁴);

q is 0 or 1;

r is 0 to 10, or 0 to 9, or 0 to 8, or 0 to 7, or 0 to 6, or 0 to 5, or0 to 4, or 0 to 3, or 0 to 2, or 0 to 1, or 1 to 10, or 1 to 9, or 1 to8, or 1 to 7, or 1 to 6, or 1 to 5, 1 to 4, or 1 to 3, or 1 to 2, or 2to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, 2 to 4, or2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8,or 8 to 10, or 8 to 9, or 9 to 10, for example, being at least 1 when Yis >N^(⊕)(R³R⁴); and

Z is SO₃ ⁻; alkyl of from 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1to 2, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 6, or 3 to 5,or 3 to 4, or 4 to 6, or 4 to 5, or 5 to 6 carbon atoms, for example;—(CHOH)_(t)(CH₂)_(u)CH₃ wherein t is 1 to 5, 1 to 4, or 1 to 3, or 1 to2, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 5, or 3 to 4, or 4 to 5, forexample, and u is 0 to 10, or 0 to 9, or 0 to 8, or 0 to 7, or 0 to 6,or 0 to 5, or 0 to 4, or 0 to 3, or 0 to 2, or 0 to 1, or 1 to 10, or 1to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, 1 to 4, or 1 to 3, or1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to5, 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7,or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, for example.

In some embodiments the pendant moiety comprises about 4 carbon atomsand about 3 heteroatoms. In some embodiments the pendant moietycomprises about 8 carbon atoms and about 7 heteroatoms. In someembodiments the pendant moiety comprises about 4 carbon atoms and about2 heteroatoms. In some embodiments the pendant moiety comprises 1 carbonatom and about 2 heteroatoms.

General Description of Assays in which the Present Compositions May BeUtilized

The following discussion is by way of illustration and not limitation.The present compositions may be employed in any assay that employs aparticle reagent. The present compositions have particular applicationto assays in which a polymer coated particle is utilized where theuncoated particle has a hydrophobic surface. The assays can be performedeither without separation (homogeneous) or with separation(heterogeneous) of any of the assay components or products.Heterogeneous assays usually involve one or more separation steps andcan be competitive or non-competitive.

Immunoassays may involve labeled or non-labeled reagents. Immunoassaysinvolving non-labeled reagents usually comprise the formation ofrelatively large complexes involving one or more antibodies. Such assaysinclude, for example, immunoprecipitin and agglutination methods andcorresponding light scattering techniques such as, e.g., nephelometryand turbidimetry, for the detection of antibody complexes. Labeledimmunoassays include chemiluminescence immunoassays, enzymeimmunoassays, fluorescence polarization immunoassays, radioimmunoassay,inhibition assay, induced luminescence, fluorescent oxygen channelingassay, and so forth.

One general group of immunoassays in which embodiments of the presentcompositions may be employed to determine the presence and/or amount ofand analyte in a sample includes immunoassays using a limitedconcentration of one of the assay reagents. Another group ofimmunoassays involves the use of an excess of one or more of theprincipal reagents. Another group of immunoassays are separation-freehomogeneous assays in which the labeled reagents modulate the labelsignal upon binding of the present composition and an analyte in thesample.

In a typical competitive heterogeneous assay, an embodiment of thepresent composition that comprises an sbp member that binds to ananalyte is contacted with a medium containing the sample suspected ofcontaining the analyte and the analyte conjugated to a label that isreactive with the sps member of the present composition or with aproduct of the activation of the sps member. Activation of the spsmember on the present composition produces a signal from the label ifthe analyte is present, which is determined by conventional techniquesand is related to the amount of the analyte in the sample.

In a typical non-competitive sandwich assay, an immune sandwich complexis formed in an assay medium. The complex comprises the analyte, an sbpmember (first sbp member) of the present compositions and a second sbpmember that binds to the analyte or to the first sbp member.Subsequently, the immune sandwich complex is detected and is related tothe amount of analyte in the sample. The immune sandwich complex isdetected by virtue of the presence in the complex of one or more of alabel of the present composition and a label of the second sbp member.

Some known assays utilize a signal producing system that employs firstand second sps members. The sps members may be related in thatactivation of one member of the sps produces a product such as, e.g.,light, which results in activation of another member of the sps.

In one approach in a sandwich assay, a first incubation of the presentcomposition is contacted with a medium containing a sample suspected ofcontaining the analyte. After a wash and separation step, the support ofthe present composition is contacted with a medium containing a secondsbp member such as, for example, an antibody for the analyte, whichcontains a label such as an enzyme, for a second incubation period. Thelabels are related in that activation of one of the labels activates theother label if the analyte is present in the medium. The support isagain washed and separated from the medium and either the medium or thesupport is examined for the presence of a signal. The presence andamount of signal is related to the presence or amount of the analyte.

In a variation of the above sandwich assay, the sample suspected ofcontaining the analyte in a suitable medium is contacted with labeledantibody for the analyte and incubated for a period of time. Then, themedium is contacted with the present composition, which comprises alabel that is related to the label of the labeled antibody as discussedabove. After an incubation period, the support is separated from themedium and washed to remove unbound reagents. The support or the mediumis examined for the presence of a signal, which is related to thepresence or amount of analyte. In another variation of the above, thesample, the present composition and the labeled antibody are combined ina medium and incubated in a single incubation step. Separation, washsteps and examination for signal are as described above.

In some embodiments of known assays, the sps members comprise asensitizer such as, for example, a photosensitizer, and achemiluminescent composition where activation of the sensitizer resultsin a product that activates the chemiluminescent composition. The secondsps member usually generates a detectable signal that relates to theamount of bound and/or unbound sps member, i.e. the amount of sps memberbound or not bound to the analyte being detected or to an agent thatreflects the amount of the analyte to be detected. In accordance withembodiments of the present invention, the present composition maycomprise one of either the sensitizer reagent or the chemiluminescentreagent.

In an embodiment of such an assay, an induced luminescence immunoassaymay be employed where the assay utilizes a composition in accordancewith the present embodiments, which includes a sensitizer or achemiluminescent compound as the sps member of the composition. Theinduced luminescence immunoassay is referred to in U.S. Pat. No.5,340,716 (Ullman), which disclosure is incorporated herein byreference. In one approach in accordance with the present embodiments,the assay uses a particle coated with a copolymer in accordance with thepresent embodiments and having associated therewith a photosensitizerand a first sbp member. The chemiluminescent reagent comprises a secondsbp member. The sbp members bind to the analyte to form a complex, orthe first sbp member binds to the second sbp member to form a complex,in relation to the presence of the analyte in the medium. If the analyteis present, the photosensitizer and the chemiluminescent compound comeinto close proximity by virtue of the binding based on the presence ofthe analyte. The photosensitizer generates singlet oxygen and activatesthe chemiluminescent reagent when the two labels are in close proximity.The activated chemiluminescent reagent subsequently produces light. Theamount of light produced is related to the amount of the complex formed,which in turn is related to the amount of analyte present.

In some embodiments of the induced luminescence assay, a photosensitizerparticle is employed that is conjugated to avidin or streptavidin. Abiotinylated sbp member that binds to the analyte is also employed. Anembodiment of the present compositions is employed where the sps memberis a chemiluminescent reagent and the sbp member binds to the analyte isemployed as part of the detection system. The reaction medium isincubated to allow the photosensitizer particles to bind to thebiotinylated sbp member by virtue of the binding between avidin andbiotin and to also allow the binding partner for the analyte that ispart of the present composition to bind to the analyte. Then, the mediumis irradiated with light to excite the photosensitizer, which is capablein its excited state of activating oxygen to a singlet state. Becausethe chemiluminescent reagent is now in close proximity to thephotosensitizer by virtue of the presence of the analyte, it isactivated by the singlet oxygen and emits luminescence. The medium isthen examined for the presence and/or the amount of luminescence orlight emitted, the presence thereof being related to the presence and/oramount of the analyte.

The concentration of the analyte that may be assayed generally variesfrom about 10⁻⁵ to about 10⁻¹⁷ M, or from about 10⁻⁶ to about 10⁻¹⁴ M.Considerations, such as whether the assay is qualitative,semi-quantitative or quantitative (relative to the amount of the analytepresent in the sample), the particular detection technique and theexpected concentration of the analyte normally determine theconcentrations of the various reagents.

The concentrations of the various reagents in the assay medium willgenerally be determined by the concentration range of interest of theanalyte, the nature of the assay, and the like. However, the finalconcentration of each of the reagents is normally determined empiricallyto optimize the sensitivity of the assay over the range. That is, avariation in concentration of analyte that is of significance shouldprovide an accurately measurable signal difference. Considerations suchas the nature of the signal producing system and the nature of theanalytes, for example, determine the concentrations of the variousreagents.

As mentioned above, the sample and reagents are provided in combinationin the medium. While the order of addition to the medium may be varied,there will be certain preferences for some embodiments of the assayformats described herein. The simplest order of addition, of course, isto add all the materials simultaneously and determine the effect thatthe assay medium has on the signal as in a homogeneous assay.Alternatively, each of the reagents, or groups of reagents, can becombined sequentially. In some embodiments, an incubation step may beinvolved subsequent to each addition as discussed above. Inheterogeneous assays, washing steps may also be employed after one ormore incubation steps.

Assay Methods Utilizing Embodiments of the Present Compositions

As mentioned above, an embodiment of the present invention is a methodof determining one or more of the presence and amount of an analyte in asample. The analyte is a substance of interest or the compound orcomposition to be detected and/or quantitated. Analytes include, forexample, drugs, metabolites, pesticides and pollutants. Representativeanalytes, by way of illustration and not limitation, include alkaloids,steroids, lactams, aminoalkylbenzenes, benzheterocyclics, purines, drugsderived from marijuana, hormones, polypeptides which includes proteins,immunosuppressants, vitamins, prostaglandins, tricyclic antidepressants,anti-neoplastics, nucleosides and nucleotides including polynucleosidesand polynucleotides, miscellaneous individual drugs which includemethadone, meprobamate, serotonin, meperidine, lidocaine, procainamide,acetylprocainamide, propranolol, griseofulvin, valproic acid,butyrophenones, antihistamines, chloramphenicol, anticholinergic drugs,and metabolites and derivatives of all of the above. Also included aremetabolites related to disease states, aminoglycosides, such asgentamicin, kanamicin, tobramycin, and amikacin, and pesticides such as,for example, polyhalogenated biphenyls, phosphate esters,thiophosphates, carbamates and polyhalogenated sulfenamides and theirmetabolites and derivatives. The term analyte also includes combinationsof two or more of polypeptides and proteins, polysaccharides and nucleicacids. Such combinations include, for example, components of bacteria,viruses, chromosomes, genes, mitochondria, nuclei and cell membranes.Protein analytes include, for example, immunoglobulins, cytokines,enzymes, hormones, cancer antigens, nutritional markers and tissuespecific antigens. Such proteins include, by way of illustration and notlimitation, protamines, histones, albumins, globulins, scleroproteins,phosphoproteins, mucoproteins, chromoproteins, lipoproteins,nucleoproteins, glycoproteins, T-cell receptors, proteoglycans, HLA,unclassified proteins, e.g., somatotropin, prolactin, insulin, pepsin,proteins found in human plasma, blood clotting factors, protein hormonessuch as, e.g., follicle-stimulating hormone, luteinizing hormone,luteotropin, prolactin, chorionic gonadotropin, tissue hormones,cytokines, cancer antigens such as, e.g., PSA, CEA, α-fetoprotein, acidphosphatase, CA19.9, CA15.3 and CA125, tissue specific antigens, suchas, e.g., alkaline phosphatase, myoglobin, CPK-MB and calcitonin, andpeptide hormones. Other polymeric materials of interest aremucopolysaccharides and polysaccharides. As indicated above, the termanalyte further includes oligonucleotide and polynucleotide analytessuch as m-RNA, r-RNA, t-RNA, DNA and DNA-RNA duplexes, for example.

The sample to be tested may be non-biological or biological.“Non-biological samples” are those that do not relate to a biologicalmaterial and include, for example, soil samples, water samples andmineral samples. The phrase “biological sample” refers to any biologicalmaterial such as, for example, body fluid, tissue and the like, which isobtained from the body of a mammal. Body fluids include, for example,whole-blood, plasma, serum, interstitial fluid, sweat, saliva, urine,semen, blister fluid, inflammatory exudates, stool, sputum, cerebralspinal fluid, tears, mucus, lymphatic fluid, vaginal mucus, and thelike. The biological tissue includes excised tissue from an organ orother body part of a host, e.g., tissue biopsies; hair and skin; and soforth.

The method comprises providing in combination in a medium the sample,which may or may not be pretreated, and an embodiment of the presentcomposition, which comprises an sbp member associated with the support.The sbp member binds to the analyte or to a second sbp member to form acomplex related to the presence of the analyte. An sbp member is one oftwo different molecules, having an area on the surface or in a cavity,which specifically binds to and is thereby defined as complementary witha particular spatial and polar organization of the other molecule. Thesbp members will usually be members of an immunological pair such asantigen-antibody, although other specific binding pairs such asbiotin-avidin, hormones-hormone receptors, enzyme-substrate, nucleicacid duplexes, IgG-protein A, polynucleotide pairs such as DNA-DNA,DNA-RNA, for example, are not immunological pairs but are includedwithin the scope of the phrase sbp member. In some embodiments,depending on the nature of the assay to be conducted as explained morefully below, other reagents are included in the medium such as, forexample, other sbp members and other sps members.

The sample can be prepared in any convenient medium. For example, thesample may be prepared in an assay medium, which is discussed more fullyhereinbelow. In some instances a pretreatment may be applied to thesample such as, for example, to lyse blood cells. Such pretreatment isusually performed in a medium that does not interfere subsequently withan assay. An aqueous medium is preferred for the pretreatment.

The sbp member is associated with the support of the composition. Insome embodiments the sbp member is covalently linked to the copolymercoated on the solid support. In some embodiments the copolymer iscovalently linked to the aldehyde bearing site of the copolymer coatingthe solid support.

An assay medium, which in some embodiments is an aqueous buffered mediumat a moderate pH, is generally one that which provides optimum assaysensitivity. The aqueous medium may be solely water or may include from0.1 to about 40 volume percent of a cosolvent such as, for example, awater miscible organic solvent, e.g., an alcohol, an ether or an amide.The pH for the medium will usually be in the range of about 4 to about11, or in the range of about 5 to about 10, or in the range of about 6.5to about 9.5, for example. The pH utilized is often the result of acompromise between optimum binding of the binding members of anyspecific binding pairs and the pH optimum for other reagents of theassay such as members of the signal producing system, for example.Various buffers may be used to achieve the desired pH and maintain thepH during the determination. Illustrative buffers include borate,phosphate, carbonate, tris, barbital, PIPES, HEPES, MES, ACES, MOPS,BICINE, and the like. The particular buffer employed is not critical,but in an individual assay one or another buffer may be preferred.

Various ancillary materials may be employed in the assay methods. Forexample, in addition to buffers the medium may comprise stabilizers forthe medium and for the reagents employed. In some embodiments, inaddition to these additives, the medium may include proteins such as,e.g., albumins; organic solvents such as, e.g., formamide; quaternaryammonium salts; polyanions such as, e.g., dextran sulfate; bindingenhancers, e.g., polyalkylene glycols; polysaccharides such as, e.g.,dextran, trehalose, or the like. The medium may also comprise agents forpreventing the formation of blood clots. Such agents are well known inthe art and include, for example, EDTA, EGTA, citrate and heparin. Themedium may also comprise one or more preservatives as are known in theart such as, for example, sodium azide, neomycin sulfate, PROCLIN® 300and Streptomycin. Any of the above materials, if employed, is present ina concentration or amount sufficient to achieve the desired effect orfunction.

As mentioned above, depending on the nature of the assay employed, themedium may further comprise one or more components such as, for example,a small molecule, an additional particle, an additional sps members andadditional binding agents such as one or more sbp members (e.g.,antibodies), which are different from those that are part of the presentcomposition. Furthermore, again depending on the nature of the assayemployed, other reagents may also be included in the initial combinationor added subsequently.

The combination is subjected to conditions for binding of the analyte tothe composition to form a complex. Such conditions may include one ormore incubation periods that may be applied to the medium at one or moreintervals including any intervals between additions of various reagentsemployed in an assay including those mentioned above, some or all ofwhich may be in the initial combination. The medium is usually incubatedat a temperature and for a time sufficient for binding of variouscomponents of the reagents and binding between complementary sbp memberssuch as, for example, an analyte and a complementary sbp member or firstand second sbp members to occur. Moderate temperatures are normallyemployed for carrying out the method and usually constant temperature,preferably, room temperature, during the period of the measurement. Insome embodiments incubation temperatures range from about 5° to about99° C., or from about 15° C. to about 70° C., or from about 20° C. toabout 45° C., for example. The time period for the incubation is about0.2 seconds to about 24 hours, or about 1 second to about 6 hours, orabout 2 seconds to about 1 hour, or about 1 to about 15 minutes, forexample. The time period depends on the temperature of the medium andthe rate of binding of the various reagents, which is determined by oneor more of the association rate constant, the concentration, the bindingconstant and dissociation rate constant, for example.

Following the above incubation periods, if any, the sps member isactivated and the amount of the complex formed between the analyte andan sbp member or between an sbp member and an sbp member that isindicative of the analyte is detected. The amount of the complex isrelated to one or both of the presence and amount of analyte in thesample. The detection of the complex is dependent on the nature of theassay being performed, the nature of the sps members, and the nature ofthe sbp members, for example.

In an embodiment, the present invention is a method of determining in asample one or more of the presence and amount of an analyte. Acombination is provided in a medium. The combination comprises thesample and a composition comprising a particle comprising a member of asignal producing system, a member of the specific binding pair thatbinds to the analyte or to a second sbp member to form a complex relatedto the presence of the analyte and a coating of a copolymer. Thecopolymer has the formula:

wherein: D is

(i) —COOR¹⁰ wherein R¹⁰ is H or alkyl of from 1 to 6, or 1 to 5, or 1 to4, or 1 to 3, or 1 to 2, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or3 to 6, or 3 to 5, or 3 to 4, or 4 to 6, or 4 to 5, or 5 to 6 carbonatoms, for example;

(ii)

wherein: w is as defined above; or

(iii) —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z

wherein:

A, R¹, n, G, R⁸, X, R², Y, m, R³, R⁴, p, q, r, Z, t and u are as definedabove;

R⁵, R⁶ and R⁷ are independently H or alkyl of from 1 to 6, or 1 to 5, or1 to 4, or 1 to 3, or 1 to 2, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to3, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 6, or 4 to 5, or 5 to 6carbon atoms, for example; and

x and y are independently 1 to about 1000, or 1 to about 800, or 1 toabout 600, or 1 to about 400, or 1 to about 200, or 1 to about 100, orabout 5 to about 1000, or about 5 to about 800, or about 5 to about 600,or about 5 to about 400, or about 5 to about 200, or about 5 to about100, or about 10 to about 1000, or about 10 to about 800, or about 10 toabout 600, or about 10 to about 400, or about 10 to about 200, or about10 to about 100, or about 50 to about 1000, or about 50 to about 800, orabout 50 to about 600, or about 50 to about 400, or about 50 to about200, or about 50 to about 100, or about 100 to about 1000, or about 100to about 800, or about 100 to about 600, or about 100 to about 400, orabout 100 to about 200, for example.

The combination is subjected to conditions for binding of the member ofthe specific binding pair to the analyte or to the second specificbinding pair member to form a complex. The member of the signalproducing system is activated and the amount of the complex is detected.The amount of the complex is related to one or more of the presence andamount of analyte in the sample.

In some embodiments of the above method, the member of the signalproducing system of the present compositions is a photosensitizer andthe combination further comprises a chemiluminescent reagent thatcomprises a particle having a chemiluminescent compound associatedtherewith and having a coating of a copolymer of the formula:

wherein: D is

(i) —COOR¹⁰ wherein R¹⁰ is as defined above;

(ii)

wherein: w is as defined above; or

(iii) —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z′

wherein:

A, R¹, n, G, R⁸, X, R², Y, m, R³, R⁴, p, q, r, t, u, R⁵, R⁶, R⁷, x and yare as defined above;

and

Z′ is SO₃ ⁻; or alkyl of from 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3,or 1 to 2, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 6, or 3to 5, or 3 to 4, or 4 to 6, or 4 to 5, or 5 to 6 carbon atoms, forexample.

In some embodiments of the above method, the member of the signalproducing system of the present compositions is a chemiluminescentcompound and the combination further comprises a photosensitizer reagentthat comprises a particle having a photosensitizer associated therewithand having a coating of a copolymer of the formula:

wherein: D is

(i) —COOR¹⁰ wherein R¹⁰ is as defined above;

(ii)

wherein: w is as defined above; or

(iii) —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z′

wherein:

A, R¹, n, G, R⁸, X, R², Y, m, R³, R⁴, p, q, r, t, u, R⁵, R⁶, R⁷, x and yare as defined above;

and

Z′ is SO₃ ⁻; or alkyl of from 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3,or 1 to 2, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 6, or 3to 5, or 3 to 4, or 4 to 6, or 4 to 5, or 5 to 6 carbon atoms, forexample.

Examination Step

In a next step of an assay method, the medium is examined for thepresence of a complex comprising the analyte. One or both of thepresence and amount of the complex indicates one or both of the presenceand amount of the analyte in the sample.

The phrase “measuring the amount of analyte” refers to the quantitative,semiquantitative and qualitative determination of the analyte. Methodsthat are quantitative, semiquantitative and qualitative, as well as allother methods for determining the analyte, are considered to be methodsof measuring the amount of the analyte. For example, a method, whichmerely detects the presence or absence of the analyte in a samplesuspected of containing the analyte, is considered to be included withinthe scope of assays in which the present compositions may be utilized.The terms “detecting” and “determining,” as well as other commonsynonyms for measuring, are contemplated within the scope of assaymethods.

In many embodiments the examination of the medium involves detection ofa signal from the medium. One or both of the presence and amount of thesignal is related to one or both of the presence and amount of theanalyte in the sample. The particular mode of detection depends on thenature of the signal producing system. As discussed above, there arenumerous methods by which a label of a signal producing system canproduce a signal detectable by external means. Activation of a signalproducing system depends on the nature of the signal producing systemmembers.

Temperatures during measurements generally range from about 10° to about70° C., or from about 20° to about 45° C., or about 20° to about 25° C.,for example. In one approach standard curves are formed using knownconcentrations of the analyte. Calibrators and other controls may alsobe used.

Luminescence or light produced from any label can be measured visually,photographically, actinometrically, spectrophotometrically or by anyother convenient means to determine the amount thereof, which is relatedto the amount of analyte in the medium. The examination for one or bothof the presence and amount of the signal also includes the detection ofthe signal, which is generally merely a step in which the signal isread. The signal is normally read using an instrument, the nature ofwhich depends on the nature of the signal. The instrument may be aspectrophotometer, fluorometer, absorption spectrometer, luminometer, orchemiluminometer, for example.

Kits Comprising Reagents for Conducting Assays

Embodiments of the present compositions and other reagents forconducting a particular assay for an analyte may be present in a kituseful for conveniently performing an assay for the determination of ananalyte. In some embodiments a kit comprises in packaged combination acomposition in accordance with the present embodiments wherein the sbpmember is an antibody for an analyte and the sps member is aphotosensitizer or a chemiluminescent compound. In some embodiments,depending on the sps member of the present compositions, the kit alsoincludes a photosensitizer or a chemiluminescent compound associatedwith an sbp member for the analyte. The kit may further include otherreagents for performing the assay, the nature of which depend upon theparticular assay format.

The reagents may each be in separate containers or various reagents canbe combined in one or more containers depending on the cross-reactivityand stability of the reagents. The kit can further include otherseparately packaged reagents for conducting an assay such as additionalsbp members, sps members and ancillary reagents, for example.

The relative amounts of the various reagents in the kits can be variedwidely to provide for concentrations of the reagents that substantiallyoptimize the reactions that need to occur during the present methods andfurther to optimize substantially the sensitivity of an assay. Underappropriate circumstances one or more of the reagents in the kit can beprovided as a dry powder, usually lyophilized, including excipients,which on dissolution will provide for a reagent solution having theappropriate concentrations for performing a method or assay utilizingembodiments of the present compositions. The kit can further include awritten description of a method as described above.

Embodiments of Copolymers

The following embodiments of copolymers are by way of illustration andnot limitation.

In some embodiments a copolymer has the formula:

wherein:

A is O or NR¹ wherein R¹ is H or alkyl of from 1 to 6, or 1 to 5, or 1to 4, or 1 to 3, or 1 to 2, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3,or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 6, or 4 to 5, or 5 to 6 carbonatoms, for example;

n is 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, 1to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7,or 2 to 6, or 2 to 5, 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, forexample; and

G′ is CHO; CH(OR⁸)₂ wherein R⁸ is alkyl of from 1 to 6, or 1 to 5, or 1to 4, or 1 to 3, or 1 to 2, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3,or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 6, or 4 to 5, or 5 to 6 carbonatoms, for example; a member of a specific binding pair;

D′ is

-   -   (i) —COOR¹⁰ wherein R¹⁰ is H or alkyl of from 1 to 6, or 1 to 5,        or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 6, or 2 to 5, or 2 to        4, or 2 to 3, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 6, or 4        to 5, or 5 to 6 carbon atoms, for example;    -   (ii)

wherein: w is 2-4, or 2-3, or 3-4, or 2, or 3, or 4; or

-   -   (iii) —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z′        wherein:

X is O or NR² wherein R² is H or alkyl of from 1 to 6, or 1 to 5, or 1to 4, or 1 to 3, or 1 to 2, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3,or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 6, or 4 to 5, or 5 to 6 carbonatoms, for example;

Y is —(CH₂O)_(m)— wherein m is 1 to 100, or 2 to 100, or 3 to 100, or 4to 100, or 1 to 90, or 1 to 80, or 1 to 70, or 1 to 60, or 1 to 50, 1 to40, or 1 to 30, or 1 to 20, or 1 to 10, or 1 to 5, or 5 to 100, or 5 to90, or 5 to 80, or 5 to 70, or 5 to 60, or 5 to 50, 5 to 40, or 5 to 30,or 5 to 20, or 5 to 10, or 10 to 100, or 10 to 90, or 10 to 80, or 10 to70, or 10 to 60, or 10 to 50, or 10 to 40, or 10 to 30, or 10 to 20, forexample; or >N^(⊕)(R³R⁴) wherein R³ and R⁴ are independently H or alkylof from 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 6,or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 6, or 3 to 5, or 3 to 4, or 4to 6, or 4 to 5, or 5 to 6 carbon atoms, for example;

p is 0 to 10, or 0 to 9, or 0 to 8, or 0 to 7, or 0 to 6, or 0 to 5, or0 to 4, or 0 to 3, or 0 to 2, or 0 to 1, or 1 to 10, or 1 to 9, or 1 to8, or 1 to 7, or 1 to 6, or 1 to 5, 1 to 4, or 1 to 3, or 1 to 2, or 2to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, 2 to 4, or2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8,or 8 to 10, or 8 to 9, or 9 to 10, for example, being at least 1 when Yis >N^(⊕)(R³R⁴);

q is 0 or 1;

r is 0 to 10, or 0 to 9, or 0 to 8, or 0 to 7, or 0 to 6, or 0 to 5, or0 to 4, or 0 to 3, or 0 to 2, or 0 to 1, or 1 to 10, or 1 to 9, or 1 to8, or 1 to 7, or 1 to 6, or 1 to 5, 1 to 4, or 1 to 3, or 1 to 2, or 2to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, 2 to 4, or2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8,or 8 to 10, or 8 to 9, or 9 to 10, for example, being at least 1 when Yis >N^(⊕)(R³R⁴); and

Z′ is SO₃ ⁻; alkyl of from 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1to 2, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 6, or 3 to 5,or 3 to 4, or 4 to 6, or 4 to 5, or 5 to 6 carbon atoms, for example;

R⁵, R⁶ and R⁷ are independently H or alkyl of from 1 to 6, or 1 to 5, or1 to 4, or 1 to 3, or 1 to 2, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to3, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 6, or 4 to 5, or 5 to 6carbon atoms, for example; and

x and y are independently 1 to about 1000, or 1 to about 800, or 1 toabout 600, or 1 to about 400, or 1 to about 200, or 1 to about 100, orabout 5 to about 1000, or about 5 to about 800, or about 5 to about 600,or about 5 to about 400, or about 5 to about 200, or about 5 to about100, or about 10 to about 1000, or about 10 to about 800, or about 10 toabout 600, or about 10 to about 400, or about 10 to about 200, or about10 to about 100, or about 50 to about 1000, or about 50 to about 800, orabout 50 to about 600, or about 50 to about 400, or about 50 to about200, or about 50 to about 100, or about 100 to about 1000, or about 100to about 800, or about 100 to about 600, or about 100 to about 400, orabout 100 to about 200, for example.

In some embodiments of the above copolymer:

A is NR¹ wherein R¹ is as defined above;

n, G, R⁸, R⁵, R⁶, R⁷, x and y are as defined above;

D′ is —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z′ wherein:

-   -   X is O;    -   Y is >N^(⊕)(R³R⁴) wherein R³ and R⁴ are as defined above;    -   p is 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1        to 5, 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2        to 8, or 2 to 7, or 2 to 6, or 2 to 5, 2 to 4, or 2 to 3, or 3        to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or        3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6,        or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to        6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7        to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, for example;    -   q is 1;    -   r is 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1        to 5, 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2        to 8, or 2 to 7, or 2 to 6, or 2 to 5, 2 to 4, or 2 to 3, or 3        to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or        3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6,        or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to        6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7        to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, for example;        and    -   Z′ is SO₃ ⁻.

In some embodiments of the above copolymer:

-   -   A is NH;    -   n is 1;    -   G is CHO; CH(OR⁸)₂ wherein R⁸ is methyl; a member of a specific        binding pair;    -   D′ is —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z′ wherein:        -   X is O;        -   Y is >N^(⊕)(R³R⁴) wherein R³ and R⁴ are both methyl;        -   p is 2;        -   q is 1;        -   r is 3;        -   Z′ is SO₃ ⁻;    -   R⁵ and R⁶ are independently H or methyl and R⁷ is H; and    -   x and y are as defined above.

In some embodiments of the above copolymer:

A is NR¹ wherein R¹ is as defined above;

n, G, R⁸,R⁵, R⁶, R⁷, x and y are as defined above;

D′ is —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z′ wherein:

-   -   X is O;    -   Y is —(CH₂O)_(m)— wherein m is as defined above;    -   p is 0;    -   q is 1;    -   r is 0; and    -   Z′ is methyl.

In some embodiments of the above copolymer:

-   -   A is NH;    -   n is 1;    -   G is CHO; CH(OR⁸)₂ wherein R⁸ is methyl; a member of a specific        binding pair;    -   D′ is —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z′ wherein:        -   X is O;        -   Y is —(CH₂O)_(m)— wherein m is as defined above;        -   p is 0;        -   q is 1;        -   r is 0;        -   Z′ is methyl;    -   R⁵ and R⁶ are independently H or methyl and R⁷ is H; and    -   x and y are as defined above.

In some embodiments of the above copolymer:

-   -   A is NR¹ wherein R¹ is H or alkyl of from 1 to 6 carbon atoms;    -   n is 1 to 10;    -   G′ is CHO; CH(OR⁸)₂ wherein R⁸ is alkyl of from 1 to 6 carbon        atoms; or a member of a specific binding pair;    -   D′ is —COOH;    -   R⁵, R⁶ and R⁷ are independently H or alkyl of from 1 to 6 carbon        atoms; and    -   x and y are independently 1 to 1000.

In some embodiments of the above copolymer:

-   -   A is NH;    -   n is 1;    -   G′ is CHO; CH(OR⁸)₂ wherein R⁸ is methyl; or a member of a        specific binding pair;    -   D′ is

-   -   wherein: w is 3;    -   R⁵ and R⁶ are independently H or methyl and R⁷ is H; and    -   x and y are independently 1 to 1000.

DEFINITIONS

The following definitions are provided for terms and phrases nototherwise specifically defined above.

The phrase “at least” as used herein means that the number of specifieditems may be equal to or greater than the number recited.

The phrase “about” as used herein means that the number recited maydiffer by plus or minus 10%; for example, “about 5” means a range of 4.5to 5.5.

The designations “first” and “second” are used solely for the purpose ofdifferentiating between two items such as, for example, “first spsmember” and “second sps member,” or “first polymerized monomer” and“second polymerized monomer” and are not meant to imply any sequence ororder or importance to one item over another or any order of addition,for example.

The following examples further describe specific embodiments of theinvention by way of illustration and not limitation and are intended todescribe and not to limit the scope of the invention. Parts andpercentages disclosed herein are by volume unless otherwise indicated.

EXAMPLES Materials

Unless indicated otherwise, reagents were purchased from Sigma-Aldrich(Milwaukee, Wis.) and used as received unless indicated otherwise.

Abbreviations:

-   -   cTnI cardiac troponin I    -   cTnI flex FLEX® cartridge of commercial cTnI assay from Siemens    -   BSA bovine serum albumin    -   LOCI luminescent oxygen channeling immunoassay    -   HEPES hydroxyethyl piperazine-ethanesulfonic acid    -   Wash buffer 50 mM HEPES, pH 8.0    -   BSA wash buffer 50 mM HEPES-1.0 mg/mL BSA, pH 8.0    -   HPMA N-2-hydroxypropylmethacrylamide    -   DMSO dimethylsulfoxide    -   AIBN azobis(isobutyronitrile)    -   PEG poly(ethylene glycol)    -   NaCNBH₃ sodium cyanoborohydride    -   MAMDMA methacrylamidoacetaldehyde dimethyl acetal    -   THF tetrahydrofuran    -   HCl hydrochloric acid    -   NaOH sodium hydroxide    -   TAPS N-tris-(hydroxymethyl)methyl-3-aminopropylsulfonic acid    -   TAPS buffer TAPS sodium salt buffer, 50 mM, pH 9.0    -   MES 2-(N-morpholino)ethanesulfonic acid    -   MES buffer 50 mM MES buffer, pH 5.0    -   STUT N,N,N′,N′-tetramethyl-O—(N-succinimidyl)-uronium        tetrafluoroborate    -   DMF dimethyl formamide    -   DMAP 4-N,N-dimethylamino-pyridine

MA.Actl methacrylamidoacetaldehyde

-   -   NHA        4,4,5,5,6,6,6-heptafluoro-1-(naphthalene-2-yl)hexane-1,3-dione    -   DPP 4,7-diphenyl-1,10-phenathroline    -   C-28 thioxene C-28 thioxene, substituted N-phenyl oxazine and        thioxene attached to 9,10-bis(phenylethynyl) anthracene (BPEA)        prepared as described in U.S. Pat. No. 6,406,667, the relevant        disclosure of which is incorporated herein by reference    -   hrs hours    -   min minutes    -   DI deionized    -   w/w weight to weight    -   rpm rounds per minute    -   mL milliliters    -   mg milligrams    -   g grams    -   mM millimolar    -   Sensibead latex particle comprising a photosensitizer dye        (bis-(trihexyl)-silicon-t-butyl-phthalocyanine) prepared using a        method analogous to that described in U.S. Pat. Nos. 6,153,442,        7,022,529, 7,229,842 and U.S. Patent Application Publication No.        20050118727A, the relevant disclosures of which are incorporated        herein by reference    -   Chemibead latex particle comprising a chemiluminescent compound        (mixture of europium NHA DPP and C-28 thioxene) prepared in a        manner such as described in U.S. Pat. No. 5,811,311, the        relevant disclosure of which is incorporated herein by        reference.

Preparation of Reagents

Synthesis of MAMDMA (FIG. 1): Methacrylic acid (9.0 g, 0.1 mole) andN-hydroxysucinimide (11.5 g, 0.1 mole) were placed in a round bottomflask and dissolved in 300 mL of THF. The solution was cooled in an icebath. Dicyclohexyl carbodiimide (21.0 g, 0.1 mole) dissolved in 50 mLTHF was added. The reaction mixture was stirred for 2 hrs in an icebath. Aminoacetaldehyde dimethyl acetal (15.0 g, 0.1 mole) andtriethylamine (15.0 g, 0.15 mole) were added. The reaction mixturesolidified to a cake and became difficult to stir due to this addition.An additional 300 mL THF was added. The reaction mixture was warmed upto room temperature and stirred for 3 days. Reaction mixture wasfiltered to remove precipitated solids. The clear solution wasconcentrated under reduced pressure. MAMDMA was obtained as a viscousliquid. Yield: 20.0 g, 90%. ¹H NMR (CDCl₃): 5.4 δ 1H, 5.6 δ 1H (doublebond protons), 3.5 δ 6H (acetal protons) 3.1 δ 1H (—CH—(OCH₃)₂), 2.5 δ2H (—CH ₂—CH—), 2.3 δ 1H (—NH—), 1.8 δ 3H (═C—CH ₃).

Procedure for polymerization of MAMDMA with a variety of hydrophilicmonomers (FIG. 2): In a round bottom flask equipped with Argon gas inletand outlet, MAMDMA (0.01M), hydrophilic monomer (0.01 mole) and AIBN(0.0001 mole, [Monomer]/[AIBN]=200) were dissolved in 30 mL DMSO. Argongas was purged through the DMSO solution at room temperature for 30 min.The flask containing monomer solution was immersed in oil bathpre-heated to 80° C. Polymerization was conducted at 80° C. for 16 hrsunder Argon purging. DMSO solution was poured into 700 mL diethyl etherto precipitate the polymer. The polymer was dissolved in 100 mL waterand concentrated to 10-15 mL using an ultrafiltration membrane ofmolecular weight cut off 5,000 daltons.

Procedure for hydrolysis of acetal groups to obtain aldehyde containingsynthetic copolymers: An aqueous solution (100 mL) containing 2-3 gcopolymer synthesized as above was taken in an Erlenmeyer flask. Tothis, 100 mL 1N HCl was added. The acidic solution was stirred for 2days at room temperature; pH of the solution was adjusted to 5.0 withthe addition of concentrated NaOH. Presence of aldehyde groups in thecopolymer was qualitatively confirmed by purpald assay (Dickinson, R.G.; Jacobsen, N. W., Chemical Communications, p. 1719 (1970). Thecopolymer solution was concentrated to 10 mL using an ultrafiltrationmembrane of molecular weight cut off 5,000 daltons. Aqueous polymersolutions (100-150 mg solids/mL) were stored at 4° C.

Procedure for preparation of Sensibead coated with aldehyde containingsynthetic copolymers: An aqueous solution of aldehyde-containingcopolymer (5 mL, pH 5.0, 100-150 mg solids/mL) was taken in a Falcontube. Solution pH was adjusted to 8.5. To this solution was added 1 mLsuspension of hydrazide-coated dyed sensibeads (25 mg/mL solids, pH9.0). The beads were coated with hydrazide by reaction of beads withhydrazine in the presence of STUT at pH 9.0 in a manner similar to thatdescribed below for the coating of chemibeads with hydrazide. Thereaction mixture was incubated at 50° C. for 72 hrs with gentle shaking.Polymer-coated beads were washed with water twice by centrifugation at15,000 rpm for 30 min at 15° C. and then were resuspended in freshwater. Polymer-coated beads were suspended in 2 mL 0.1M acetate bufferpH 5.0. To this, streptavidin (7.0 mg) was added and incubated at roomtemperature for 30 min. To this, NaCNBH₃ (40 mg) was added and incubatedat 37° C. for 72 hrs with gentle shaking. Streptavidin-coated beads(sensibeads) were washed with BSA wash buffer twice by centrifugation at15,000 rpm for 30 min at 15° C. and were resuspended in the buffer.Washed sensibeads were suspended by sonication for 2 minutes at 4° C.using a probe sonicator. Solid contents of sensibeads were determinedand adjusted to 10 mg/mL. The streptavidin number per particle wasdetermined from the depletion of fluorescence when differentconcentrations of particles were incubated with a fixed concentration ofthe biotin-fluorescein conjugate. Sensibeads were stored at 4° C. at 10mg/mL concentration until further use. The results are set forth inTable 1.

TABLE 1 Sensibead No. Streptavidin No. SB 1 1150 Poly(HPMA-co-MA-Actl)(1:1) SB 2 5313 Poly(HPMA-co-MA-Actl) (1:2) SB 3 3713Poly(HPMA-co-MA-Actl) (1:4) SB 4 6274 Poly(sulfobetaine-co-MA-Actl)(1:1) SB 5 1477 Poly(MPEG₁₁₀₀-MA-co-MA-Actl) (1:1) SB 6 3211Poly(AA-co-MA-Actl) (1:1) SB 7 2188 Poly(MPEG₃₀₀-MA-co-MA-Actl) (1:1) SB8 ND Poly(NVP-co-MA-Actl) (1:1)

Preparation of Chemibeads Coated with Synthetic Copolymer:

Step 1: Preparation of hydrazide-coated dyed chemibead: Chemibeadsuspension (15 mL, 1.046 g particles) was taken in a round bottom flask.To this, 37 mL 300 mM TAPS buffer pH 9.0 was added to make 20 mg/mLparticle suspension. Hydrazine (0.1 mL) was added and pH electrode wasdipped in the particle suspension and stirred at room temperature. Tothis, 100 mg STUT (freshly dissolved in 1 mL DMF) was added followed byaddition of 0.25 mL DMF solution of DMAP (100 mg/mL). The reactionmixture was stirred for 10 min and the pH was adjusted to 9.0 withaddition of 5 N NaOH. STUT and DMAP addition was repeated 4 times andthe particle suspension was stirred for an additional 1 hr.Hydrazide-coated, dyed chemibeads were washed twice with 1 mM TAPSbuffer, pH 9.0, by centrifugation at 15,000 rpm, 30 min and 10° C. andwere resuspended in the buffer. Washed particles were suspended in 17 mL1 mM TAPS buffer pH 9.0. Solids content was 41.23 mg/mL.

Step 2: Preparation of poly(HPMA-co-MA-Actl (1:2)) coated dyedchemibead: Hydrazide-coated chemibead suspension (0.6 mL, 25 mg beads)from above was taken in a 15 mL Falcon tube. Poly(HPMA-co-MA-Actl (1:2,FIG. 2) (3 mL, pH 5.0, solids content 24.7 mg/mL) was taken in anothertube and the pH was adjusted to 8.8. Polymer solution of pH 8.8 andhydrazide-coated bead suspension of pH 9.0 were mixed together andincubated at 37° C. for 50 hrs with gentle shaking. Polymer coated beads(CB 2) were washed twice with 50 mM MES buffer, pH 6.0, bycentrifugation at 15,000 rpm, 30 min, 10° C. and were resuspended in theMES buffer. Washed beads were resuspended in 0.25 mL MES buffer bysonication using a probe sonicator.

Step 3: Preparation of capture antibody-coated chemibead: cTnI captureantibody was buffer exchanged with 10 mM PO₄-300 mM NaCl, pH 7.0containing 0.2% TWEEN 20®. Concentration of buffer exchanged antibodywas 7.7 mg/mL. cTnI capture antibody solution (1 mL, 7.7 mg antibody),poly(HPMA-co-MA-Actl (1:2) coated chemibeads (0.25 mL, 25 mg beads),acetic acid (0.4 μL) were mixed together at 4° C. The mixture had pH5.7. NaCNBH₃ (12 μL, 25 mg/mL) was added and the suspension incubated at4° C. for 16 hrs with gentle shaking. The particles were then incubatedat 37° C. for 24 hrs. Antibody-coated chemibeads were washed twice withwash buffer (pH 8.0) by centrifugation at 15,000 rpm, 30 min, 10° C. andwere resuspended in the wash buffer. Washed particles were resuspendedin 1 mL BSA wash buffer (pH 8.0) by sonication with a probe sonicator.Solids content was 13.37 mg/mL.

Molecular weight characterization of aldehyde-containing syntheticcopolymers: Polymers were characterized by size exclusion chromatographycoupled with light scattering. Data obtained are summarized in Table 2.All synthetic polymers were synthesized by conventional free radicalpolymerization which is known to produce polymers with a largepolydispersity. Data in Table 2 also show that aldehyde-containingcopolymers were polydispersed. Peaks mentioned in Table 2 for polymersare not strictly bimodal but rather deconvoluted from a broad Gaussianpeak present for each polymer as determined using the software Astra(Wyatt Technology Corporation, Santa Barbara Calif.) from lightscattering detector. Molecular weight of predominant peaks in polymersamples varied from 25 kDa to 3300 kDa depending on individual monomerstructure and its polymerizability.

TABLE 2 Molecular weight characterization of aldehyde-containingcopolymers Peak 1 Peak 2 MW (Da) MW (Da) SB PolydispersityPolydispersity No. Polymer index % Mass index % Mass 1Poly(HPMA-co-MA-Actl) (1:1) 120,200 40,330 1.13 1.10 29.6 73.1 2Poly(HPMA-co-MA-Actl) (1:2) 68,070 26,940 1.08 1.09 27.1 72.9 3Poly(HPMA-co-MA-Actl) (1:4) Not determined. Polymer gelled beforeperforming analysis. 4 Poly 467,400 89,560 (sulfobetaine-co-MA-Actl)1.44 1.09 (1:1) 64.5 35.5 5 Poly 3,353,000 — (MPEG₁₁₀₀-MA-co-MA-Actl)2.12 — (1:1) 100 — 6 Poly(AA-co-MA-Actl) (1:1) 47,980 14,200 1.13 1.1721.4 78.6 7 Poly 1,447,000 — (MPEG₃₀₀-MA-co-MA-Actl) 1.062 — (1:1) 100 —8 Poly(NVP-co-MA-Actl) (1:1) 399,200 38,340 2.18 1.09 26.8 73.2

Assays

General procedure for cTnI assay using Sensibeads coated withembodiments of the present copolymers: All assays were performed on aDIMENSION® VISTA® instrument (Siemens Healthcare Diagnostics Inc.,Newark DE) (Siemens). Briefly, cover on well #8 of a commercial cTnIflex cartridge (Siemens Healthcare Diagnostics catalog #K6421) waspunctured. The commercial cTnI product utilizes LOCI technology andreagents. Dextran-coated sensibead suspension of the commercial productwas aspirated out and the copolymer-coated sensibead suspension (0.7 mL,1.5 mg sensibead/mL BSA wash buffer) was added to the cleaned well. ThecTnI assay was then run using calibrators as samples according to themanufacturer's instructions supplied with the product. No optimizationwas carried out either for instrument parameters, buffer formulations,or concentration of reagents employed. Results of assays are summarizedbelow in Table 3 (SB 1), Table 4 (SB 2), Table 5 (SB 3), Table 6 (SB 4),Table 7 (SB 5), Table 8 (SB 6), Table 9 (SB 7) and Table 10 (SB 8).

TABLE 3 Assays using SB 1 LOCI Signal (kcounts) Cali- brator ng/mL Rep 1Rep 2 Rep 3 Mean SD % CV L1 0.00 6.67 6.69 6.61 6.66 0.04 0.6% L2 0.4819.96 20.15 19.68 19.93 0.24 1.2% L3 4.30 185.09 182.21 182.22 183.171.66 0.9% L4 8.40 418.64 421.13 423.99 421.25 2.68 0.6% L5 20.70 1339.861313.21 1334.42 1329.16 14.08 1.1% L6 42.90 2848.55 2825.92 2854.272842.91 14.99 0.5%

TABLE 4 Assays using SB 2 LOCI Signal (kcounts) Cali- brator ng/mL Rep 1Rep 2 Rep 3 Mean SD % CV L1 0.00 3.46 3.46 3.31 3.41 0.09 2.5% L2 0.4816.18 16.17 16.14 16.16 0.02 0.1% L3 4.30 168.59 168.17 168.73 168.500.29 0.2% L4 8.40 381.76 383.77 387.01 384.18 2.65 0.7% L5 20.70 1191.471188.01 1193.07 1190.85 2.59 0.2% L6 42.90 2580.13 2556.88 2557.882564.96 13.14 0.5%

TABLE 5 Assays using SB 3 LOCI Signal (kcounts) Cali- brator ng/mL Rep 1Rep 2 Rep 3 Mean SD % CV L1 0.00 20.6 20.47 20.24 20.44 0.18 0.9% L20.48 57.25 61.08 55.35 57.89 2.92 5.0% L3 4.30 427.97 417.57 419.15421.56 5.60 1.3% L4 8.40 875.66 870.74 879.83 875.41 4.55 0.5% L5 20.702153.91 2157.21 2162.87 2158.00 4.53 0.2% L6 42.90 3655.09 3603.633639.14 3632.62 26.34 0.7%

TABLE 6 Assays using SB 4 LOCI Signal (kcounts) Cali- brator ng/mL Rep 1Rep 2 Rep 3 Mean SD % CV L1 0.00 3.5 3.12 3.09 3.24 0.23 7.1% L2 0.488.87 8.72 9.02 8.87 0.15 1.7% L3 4.30 74.68 73.61 73.33 73.87 0.71 1.0%L4 8.40 167.6 170.81 167.73 168.71 1.82 1.1% L5 20.70 538.68 533.22538.89 536.93 3.21 0.6% L6 42.90 1254.19 1241.71 1232.78 1242.89 10.750.9%

TABLE 7 Assays using SB 5 LOCI Signal (kcounts) Cali- brator ng/mL Rep 1Rep 2 Rep 3 Mean SD % CV L1 0.00 5.96 5.98 5.7 5.88 0.16 2.7% L2 0.486.2 6.35 6.2 6.25 0.09 1.4% L3 4.30 12.79 12.52 12.42 12.58 0.19 1.5% L48.40 20.32 20.72 20.52 20.52 0.20 1.0% L5 20.70 52.09 52.97 52.27 52.440.46 0.9% L6 42.90 121.84 121.2 121.73 121.59 0.34 0.3%

TABLE 8 Assays using SB 6 LOCI Signal (kcounts) Cali- brator ng/mL Rep 1Rep 2 Rep 3 Mean SD % CV L1 0.00 19.59 19.79 18.24 19.21 0.84 4.4% L20.48 26.82 24.69 26.71 26.07 1.20 4.6% L3 4.30 81.16 83.34 79.84 81.451.77 2.2% L4 8.40 153.17 142.64 144.11 146.64 5.70 3.9% L5 20.70 367.98370.68 329.42 356.03 23.08 6.5% L6 42.90 685 625.57 677.17 662.58 32.294.9%

TABLE 9 Assays using SB 7 LOCI Signal (kcounts) Cali- brator ng/mL Rep 1Rep 2 Rep 3 Mean SD % CV L1 0.00 4.25 4.29 4.72 4.42 0.26 5.9% L2 0.485.37 5.73 5.51 5.54 0.18 3.3% L3 4.30 19.8 19.47 19.91 19.73 0.23 1.2%L4 8.40 40.74 40.55 41.37 40.89 0.43 1.0% L5 20.70 126.38 125.28 123.42125.03 1.50 1.2% L6 42.90 317.63 316.73 319.54 317.97 1.43 0.5%

TABLE 10 Assays using SB 8 LOCI Signal (kcounts) Cali- brator ng/mL Rep1 Rep 2 Rep 3 Mean SD % CV L1 0.00 3.55 3.45 3.8 3.60 0.18 5.0% L2 0.489.06 9.07 8.61 8.91 0.26 2.9% L3 4.30 66.07 65.5 66.3 65.96 0.41 0.6% L48.40 144.2 146 145.83 145.34 0.99 0.7% L5 20.70 386.51 390.04 383.37386.64 3.34 0.9% L6 42.90 727.9 740.82 730 732.91 6.93 0.9%

General procedure for cTnI assay using SB 2 and CB 2: All assays wereperformed on a DIMENSION® VISTA® instrument (Siemens HealthcareDiagnostics Inc., Newark Del.) (Siemens). Briefly, cover on well #8 of acommercial cTnI flex cartridge was punctured. Dextran-coated sensibeadsuspension was aspirated out and SB 2 suspension (0.7 mL, 1.5 mg SB 2/mLBSA wash buffer) was added into the cleaned well. In addition,chemibeads of the commercial cTnI product were replaced with CB 2 (0.9mL, 0.19 mg CB 2/mL BSA wash buffer). The cTnI assay was then run usingcalibrators as samples according to the manufacturer's instructionssupplied with the product. No optimization was carried out either forinstrument parameters, buffer formulations, or concentration of reagentsemployed. Results of assays are summarized below in Table 11.

TABLE 11 Assays using SB 2 and CB 2 LOCI Signal (kcounts) Cali- bratorng/mL Rep 1 Rep 2 Rep 3 Mean SD % CV L1 0.00 15.84 15.86 16.11 15.940.15 0.9% L2 0.48 17.8 17.3 17.91 17.67 0.33 1.8% L3 4.30 39.62 40.239.35 39.72 0.43 1.1% L4 8.40 70.01 68.5 66.93 68.48 1.54 2.2% L5 20.70176.42 177.26 178.42 177.37 1.00 0.6% L6 42.90 370.14 379.3 381.45376.96 6.01 1.6%

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. Furthermore, the foregoing description,for purposes of explanation, used specific nomenclature to provide athorough understanding of the invention. However, it will be apparent toone skilled in the art that the specific details are not required inorder to practice the invention. Thus, the foregoing descriptions ofspecific embodiments of the present invention are presented for purposesof illustration and description; they are not intended to be exhaustiveor to limit the invention to the precise forms disclosed. Manymodifications and variations are possible in view of the aboveteachings. The embodiments were chosen and described in order to explainthe principles of the invention and its practical applications and tothereby enable others skilled in the art to utilize the invention.

1. A composition for use as an assay reagent, the compositioncomprising: a solid support comprising a member of a signal producingsystem and a coating of a synthetic copolymer wherein the syntheticcopolymer comprises a first copolymerized monomer comprising a pendantmoiety comprising a reactive functionality or a derivatized reactivefunctionality and a second copolymerized monomer comprising a pendantmoiety comprising at least 1 carbon atom and at least 2 heteroatoms. 2.The composition according to claim 1 wherein the solid support is aparticle.
 3. The composition according to claim 2 wherein the particleis a latex particle.
 4. The composition according to claim 1 wherein themember of the signal producing system is selected from the groupconsisting of sensitizers and chemiluminescent compounds.
 5. Thecomposition according to claim 1 wherein the copolymer comprises anpolyethylenic backbone comprising (i) the pendant moiety comprising areactive functionality or a derivatized reactive functionality and (ii)the pendant moiety comprising at least 1 carbon atom and at least 2heteroatoms.
 6. The composition according to claim 1 wherein the pendantmoiety of the first copolymerized monomer is —C(O)-A-(CH₂)_(n)-G whereinA is O or NR¹ wherein R¹ is H or alkyl of from 1 to 6 carbon atoms and nis 1 to 10 and wherein G is CHO; CH(OR⁸)₂ wherein R⁸ is alkyl of from 1to 6 carbon atoms; COOH or a derivative thereof; NR¹ wherein R¹ is H oralkyl of from 1 to 6 carbon atoms; OH; or a member of a specific bindingpair.
 7. The composition according to claim 1 wherein the pendant moietyof the second copolymerized monomer comprising at least 1 carbon atomand at least 2 heteroatoms comprises one or more groups selected fromthe groups consisting of hydroxyl groups, carboxyl groups, amine groups,amide groups, ester groups, polyoxyethylene groups, polyoxypropylenegroups, sulfate groups, sulfite groups, phosphate groups, phosphitegroups, phosphatidylcholine groups, betaine groups and sulfobetainegroups.
 8. The composition according to claim 1 wherein the pendantmoiety comprising at least 1 carbon atoms and at least 2 heteroatoms hasthe formula (i) —COOR¹⁰ wherein R¹⁰ is H or alkyl of from 1 to 6 carbonatoms, (ii)

wherein: w is 2-4; or (iii) —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Zwherein: X is O or NR² wherein R² is H or alkyl of from 1 to 6 carbonatoms; Y is —(CH₂O)_(m)— wherein m is 1 to 1500; or >N^(⊕)(R³R⁴) whereinR³ and R⁴ are independently H or alkyl of from 1 to 6 carbon atoms; p is0 to 10, being at least 1 when Y is >N^(⊕)(R³R⁴); q is 0 or 1; r is 0 to10, being at least 1 when Y is >N^(⊕)(R³R⁴); and Z is SO₃ ⁻; alkyl offrom 1 to 6 carbon atoms; —(CHOH)_(t)(CH₂)_(u)CH₃ wherein t is 1 to 5and u is 0 to
 10. 9. The composition according to claim 1 wherein amember of a specific binding pair is associated with the solid support.10. A method of determining in a sample the presence and/or amount of ananalyte, the method comprising: (a) providing in combination in amedium: (i) the sample, and (ii) the composition according to claim 9wherein the member of the specific binding pair (sbp) binds to theanalyte or to a second sbp member to form a complex related to thepresence of the analyte, (b) subjecting the combination to conditionsfor binding of the analyte to the composition to form a complex, and (c)activating the member of the signal producing system and detecting theamount of the complex, the amount of the complex being related to thepresence and/or amount of analyte in the sample.
 11. A method ofdetermining in a sample the presence and/or amount of an analyte, themethod comprising: (a) providing in combination in a medium: (i) thesample, and (ii) a composition comprising: a particle comprising: (A) amember of a signal producing system, (B) a member of the specificbinding pair that binds to the analyte or to a second sbp member to forma complex related to the presence of the analyte and (C) a coating of acopolymer of the formula:

wherein: A is O or NR¹ wherein R¹ is H or alkyl of from 1 to 6 carbonatoms; n is 1 to 10; G is CHO; CH(OR⁸)₂ wherein R⁸ is alkyl of from 1 to6 carbon atoms; COOH or a derivative thereof; NR¹ wherein R¹ is H oralkyl of from 1 to 6 carbon atoms; OH; or a member of a specific bindingpair; D is (i) —COOR¹⁰ wherein R¹⁰ is H or alkyl of from 1 to 6 carbonatoms; (ii)

wherein: w is 2-4; or (iii) —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)-—Zwherein: X is O or NR² wherein R² is H or alkyl of from 1 to 6 carbonatoms; Y is —(CH₂O)_(m)— wherein m is 1 to 1500; or >N^(⊕)(R³R⁴) whereinR³ and R⁴ are independently H or alkyl of from 1 to 6 carbon atoms; p is0 to 10, being at least 1 when Y is >N^(⊕)(R³R⁴); q is 0 or 1; r is 0 to10, being at least 1 when Y is >N^(⊕)(R³R⁴); and Z is SO₃ ⁻; alkyl offrom 1 to 6 carbon atoms; —(CHOH)_(t)(CH₂)_(u)CH₃ wherein t is 1 to 5and u is 0 to 10; R⁵, R⁶ and R⁷ are independently H or alkyl of from 1to 6 carbon atoms; and x and y are independently 1 to 1000; (b)subjecting the combination to conditions for binding of the member ofthe specific binding pair to the analyte or to the second specificbinding pair member to form a complex; and (c) activating the member ofthe signal producing system and detecting the amount of the complex, theamount of the complex being related to the presence and/or amount ofanalyte in the sample.
 12. The method according to claim 11 wherein themember of the signal producing system is selected from the groupconsisting of sensitizers and chemiluminescent compounds.
 13. The methodaccording to claim 11 wherein the member of the signal producing systemis incorporated into the particle.
 14. The method according to claim 11wherein the member of the signal producing system is a photosensitizerand the combination further comprises a chemiluminescent reagent orwherein the member of the signal producing system is a chemiluminescentcompound and the combination further comprises a photosensitizerreagent.
 15. The method according to claim 14 wherein thechemiluminescent reagent or the photosensitizer reagent comprises: aparticle comprising a coating of a copolymer of the formula:

wherein: A is O or NR¹ wherein R¹ is H or alkyl of from 1 to 6 carbonatoms; n is 1 to 10; G is CHO; CH(OR⁸)₂ wherein R⁸ is alkyl of from 1 to6 carbon atoms; COOH or a derivative thereof; NR¹ wherein R¹ is H oralkyl of from 1 to 6 carbon atoms; OH; or a member of a specific bindingpair; D is (i) —COOR¹⁰ wherein R¹⁰ is H or alkyl of from 1 to 6 carbonatoms; (ii)

wherein: w is 2-4; or (iii) —C(O)—X—(CH₂)_(p)—(Y)_(p)—(CH₂)_(r)—Zwherein: X is O or NR² wherein R² is H or alkyl of from 1 to 6 carbonatoms; Y is —(CH₂O)_(m)— wherein m is 1 to 1500; or >N^(⊕)(R³R⁴) whereinR³ and R⁴ are independently H or alkyl of from 1 to 6 carbon atoms; p is0 to 10, being at least 1 when Y is >N^(⊕)(R³R⁴); q is 0 or 1; r is 0 to10, being at least 1 when Y is >N^(⊕)(R³R⁴); and Z is SO₃ ⁻; alkyl offrom 1 to 6 carbon atoms; —(CHOH)_(r)(CH₂)_(u)CH₃ wherein t is 1 to 5and u is 0 to 10; R⁵, R⁶ and R⁷ are independently H or alkyl of from 1to 6 carbon atoms; and x and y are independently 1 to
 1000. 16. Acopolymer of the formula:

wherein: A is O or NR¹ wherein R¹ is H or alkyl of from 1 to 6 carbonatoms; n is 1 to 10; G′ is CHO; CH(OR⁸)₂ wherein R⁸ is alkyl of from 1to 6 carbon atoms; a member of a specific binding pair; D′ is (i)—COOR¹⁰ wherein R¹⁰ is H or alkyl of from 1 to 6 carbon atoms; (ii)

wherein: w is 2-4; or (iii) —C(O)—X—(CH₂)_(p)—(Y)_(p)—(CH₂)_(r)—Z′wherein: X is O or NR² wherein R² is H or alkyl of from 1 to 6 carbonatoms; Y is —(CH₂O)_(m)— wherein m is 1 to 1500, or >N^(⊕)(R³R⁴) whereinR³ and R⁴ are independently H or alkyl of from 1 to 6 carbon atoms; p is0 to 10, being at least 1 when Y is >N^(⊕)(R³R⁴); q is 0 or 1; r is 0 to10, being at least 1 when Y is >N^(⊕)(R³R⁴); and Z′ is SO₃ ⁻; or alkylof from 1 to 6 carbon atoms; R⁵, R⁶ and R⁷ are independently H or alkylof from 1 to 6 carbon atoms; and x and y are independently 1 to about1000.
 17. The copolymer according to claim 16 wherein: A is NR¹ whereinR¹ is H or alkyl of from 1 to 6 carbon atoms; n is 1 to 10; G′ is CHO;CH(OR⁸)₂ wherein R⁸ is alkyl of from 1 to 6 carbon atoms; or a member ofa specific binding pair; D′ is —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z′wherein: X is O; Y is >N^(⊕)(R³R⁴) wherein R³ and R⁴ are independently Hor alkyl of from 1 to 6 carbon atoms; p is 1 to 10; q is 1; r is 1 to10; Z′ is SO₃ ⁻; R⁵, R⁶ and R⁷ are independently H or alkyl of from 1 to6 carbon atoms; and x and y are independently 1 to
 1000. 18. Thecopolymer according to claim 17 wherein: A is NH; n is 1; G′ is CHO;CH(OR⁸)₂ wherein R⁸ is methyl; or a member of a specific binding pair;D′ is —C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z′ wherein: X is O; Y is>N^(⊕)(R³R⁴) wherein R³ and R⁴ are both methyl; p is 2; q is 1; r is 3;Z′ is SO₃ ⁻; R⁵ and R⁶ are independently H or methyl and R⁷ is H; and xand y are independently 1 to
 1000. 19. The copolymer according to claim16 wherein: A is NR¹ wherein R¹ is H or alkyl of from 1 to 6 carbonatoms; n is 1 to 10; G′ is CHO; CH(OR⁸)₂ wherein R⁸ is alkyl of from 1to 6 carbon atoms; or a member of a specific binding pair; D′ is—C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z′ wherein: X is O; Y is—(CH₂O)_(m)— wherein m is 1 to 1500; p is 0; q is 1; r is 0; Z′ ismethyl; R⁵, R⁶ and R⁷ are independently H or alkyl of from 1 to 6 carbonatoms; and x and y are independently 1 to
 1000. 20. The copolymeraccording to claim 19 wherein: A is NH; n is 1; G′ is CHO; CH(OR⁸)₂wherein R⁸ is methyl; or a member of a specific binding pair; D′ is—C(O)—X—(CH₂)_(p)—(Y)_(q)—(CH₂)_(r)—Z′ wherein: X is O; Y is—(CH₂O)_(m)— wherein m is 1100 or 300; p is 0; q is 1; r is 0; Z′ ismethyl; R⁵ and R⁶ are independently H or methyl and R⁷ is H; and x and yare independently 1 to 1000.